Green Tea Polyphenol Epigallocatechin-3-Gallate Inhibits TNF-a-Induced Production of Monocyte Chemoattractant Protein-1 in Human Umbilical Vein Endothelial Cells

Green tea, produced from the leaves of the plant Camellia sinensis, is widely consumed beverage throughout the world. Epidemiological studies and associated meta-analyses have reported that daily consumption of green tea is associated with many health benefits, such as a reduced risk of coronary artery disease (CAD) [1,2]. Green tea contains many biologically active polyphenolic flavonoid, commonly known as catechins, which is a family that includes (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epigallocatechin-3-gallate (EGCG) [3,4]. As the principal constituent, EGCG has a variety of biological and pharmacological properties and has received attention as a prospective dietary intervention in cardiovascular diseases (CVD) [4].

Dysregulated expression of molecules involved in inflammation which results from endothelial dysfunction is associated with the initiation and the progression of CVD [5]. Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, can induce the activation of the vascular endothelium and plays a pivotal role in vascular disease development by directly promoting inflammation, oxidative stress, vascular remodeling and atherogenesis [6,7]. It has been suggested that many cardiovascular risk factors contribute to the pathogenesis of endothelial dysfunction through the modulation of TNF-α signaling [6]. Monocyte chemotactic protein-1 (MCP-1) is a chemoattractant and can be produced by many cell types, including endothelial, epithelial, fibroblasts, smooth muscle cells and macrophages [8,9,10]. In patients at risk for CAD, elevated MCP-1 serum levels have been suggested as a direct marker of the inflammatory activity [10,11].

The 67-kDa laminin receptor (67LR) is a non-integrin cell-surface receptor with high affinity for laminin; it plays a key role in tumor invasion and metastasis [12]. Recently, 67LR has been identified as a cell surface EGCG receptor that mediates the anti-inflammatory activity of EGCG. In macrophages, 67LR was shown to be involved in the inhibitory effect of EGCG on the LPS-induced TLR4 signaling and peptidoglycan-induced TLR2 signaling pathway [13,14]. It was also shown to be involved in the inhibitory effect of EGCG on the TLR4 signaling pathway in dendritic cells [15]. Recently, it has been demonstrated that 67LR as a cell-surface EGCG receptor mediates the anti-inflammatory action of EGCG in endotoxin-stimulated human cerebral microvascular endothelial cells (hCMECs) [16]. Nonetheless, the relationship between 67LR and anti-inflammatory activity of EGCG in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs) has not yet been established.

In the present study, we tried to investigate the effects of EGCG on HUVECs exposed to TNF-α by evaluating the expression of MCP-1. Furthermore, the mechanisms through which EGCG exerts its action were investigated.

The HUVECs-derived EA.hy 926 cells were gifted by Atherosclerosis Research Lab of Nanjing Medical University. They obtained this cell line from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (CBTCCCAS, Shanghai, China). Cells were maintained in RPMI 1640 medium (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) without antibiotic in a humidified atmosphere at 37°C in 5% CO2 as previously described [17]. HUVECs at passage 5-7 were used for the experiments. The culture medium was changed to RPMI 1640 supplemented with 0.1% FBS for an additional 12 h before the experiments. HUVECs were treated with different concentrations of EGCG (Sigma-Aldrich St. Louis, MO, USA) (10, 25, and 50 μM) for 1 h prior to TNF-α (10 ng/ml) treatment and further incubated together for different time periods. For the blockage of 67LR, cells were incubated with anti-67LR Ab (clone MluC5; 20 μg/ml; NeoMarkers, Fermont, CA, USA) or isotype-matched control mouse IgM (20 μg/ml) for 1 h before the treatment of EGCG.

HUVECs were seeded in 6-well microplates and grown to confluence. They were pretreated with different concentrations of EGCG (10, 25, and 50 µM) for 1 h and stimulated by TNF-α (10 ng/ml) for 24 h. After treatment, supernatants of cell cultures were collected and concentrated using centrifugal filter units to remove cellular debris; the MCP-1 levels were quantified using ELISA (Boster Biotech, Wuhan, China). Assays were performed according to the manufacturer's instructions.

Total cellular RNA was extracted using Trizol reagent (Invitrogen Life Technologies) according to the manufacturer's instructions and reverse transcribed into cDNA using PrimeScript RT reagent kit Perfect Real (Takara, Dalian, China). First the cDNA was quantified in a 1:10 dilution on a spectrophotometer. MCP-1 mRNA expression was determined by RT-qPCR on an ABI PRISM 7500 real-time PCR apparatus (Applied Biosystems, USA) with SYBR Premix Ex Taq TM II. The conditions for quantitative real-time PCR were: 95˚C for 30 s, then 40 cycles at 95˚C for 5 s, and 60˚C for 34 s, and a final extension of 95°C for15 s, 60°C for 1 min and 95°C for 15 s. The mRNA level for MCP-1 of each sample was normalized to Ct values of the GAPDH amplified from the same sample, ΔCt=Ct_MCP-1 - Ct_GAPDH and the 2^-ΔΔCt method was used to calculate gene expression change. Samples were measured in triplicates to ensure the reproducibility of the results. 67LR mRNA expression was evaluated by RT-PCR. cDNA was subsequently amplified in 50μl reaction volumes containing 0.4 μmol/l of each 67LR primer and 1.25 U Taq DNA polymerase (TaKaRa, Dalian, China). The amplification conditions were pre-denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 56°C for 30 s and 72°C for 40 s, and a final extension of 72°C for 7 min. PCR products were electrophoresed on a 1% agarose gel containing ethidium bromide and then visualized under UV light. The primer sequences are shown in Table 1.

The viability of HUVECs was determined using the Cell Counting Kit-8 Assay Kit (Beyotime Institute of Biotechnology, China), as described previously [18]. Briefly, 5 × 10³ cells/well were seeded in a 96-well plate and cultured to confluence. Subsequently, cells were treated with EGCG at increasing concentrations (0, 10, 25, and 50 μM) for 24 h. After 10 μl CCK-8 solution was add to each well, the absorbance at 450 nm was measured 4 h after incubation in the dark.

Cells were harvested and lysed, and then protein concentration was measured using Pierce BCA protein assay reagent (Thermo-Fisher Scientific, Rockford, Ill., USA). Cell lysates were electrophoresed through 12% polyacrylamide gels and transblotted onto polyvinylidene difluoride (PVDF) membranes in a semidry transfer system (Amersham Pharmacia Biotech AB, Uppsala, Sweden). After blocking with 5% BSA in TBST buffer (10 m M Tris, pH 7.5, 150 m M NaCl, and 0.05% Tween-20) for 2 h at room temperature, the membrane was probed with rabbit polyclonal antibody to P-p65 (1: 5,000; Santa Cruz, Calif., USA), mouse monoclonal antibody to IκBα (1: 1000; Cell Signaling Technology, MA., USA), and mouse monoclonal antibody to β-actin (1: 1,000; Santa Cruz, Calif., USA) overnight at 4 °C. The antibodies were detected using 1: 5,000 goat anti-rabbit (Biosynthesis, Beijing, China) and 1: 2,000 goat anti-mouse (Santa Cruz, Calif., USA) horseradish peroxidase-conjugated antibodies for 1 h at room temperature. Membranes were developed with enhanced chemiluminescence detection (ECL, Amersham, Bucks, UK). The immunoreactivity was quantitated using Image J software.

HUVECs were seeded in six-well plates (1.5×10⁵ cells per well) overnight and cotransfected with NF-κB reporter plasmid (Promega) and pRL-TK (Promega) as an internal control. Transfection complexes were prepared by mixing FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) and DNA in a 3:1 ratio in serum-free medium (3 μl FuGENE HD to 1μg DNA per plasmid per well). The complexes were incubated for 15 min before added to cells. Six hours after the transient transfection, the cells were serum-starved for 12 h and incubated with or without EGCG (10, 25, and 50μM) for 1 h, then subjected to TNF-αstimulation for an additional 6 h. Firefly and Renilla luciferase activities in cell extracts were assayed using the Dual-Luciferase Reporter Assay System (Promega). The relative luciferase activity represents the ratio of the activity of firefly to control Renilla luciferase activity [19].

We used monocyte adhesion assay to evaluate the physiological consequences of the inhibitory effect of EGCG on induction of MCP-1 [19,20]. Briefly, HUVECs were seeded on 2% gelatin-coated six-well plates and cultured to confluence. The cells were pre-treated with EGCG (10, 25, 50 μM) for 1 h and stimulated with TNF-α(10 ng/mL) for 6 h. U937 cells (CBTCCCAS, Shanghai, China) were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100μg /ml streptomycin. After washing three times with serum-free RPMI 1640 medium, approximately 1 mL of U937 cells (20,000 cells/mL) was added to the wells and incubated for 20 min at 37°C to allow the adhesion. The plates were then carefully washed out three times with serum-free RPMI 1640 medium to remove non-adherent cells. The adherent monocytes were counted in five randomly selected optical fields in each well under a microscope (Olympus).

Values are expressed as mean ± standard error. Statistical significance between groups was assessed by using one-way ANOVA followed by Fisher's exact test. It was considered statistically significant when the P-value was less than 0.05.

The results in Fig. 1 showed that MCP-1 in the culture supernatant was markedly elevated after the exposure of HUVECs to 10ng/ml TNF-α for 24 h. However, pretreatment of the cells with different concentrations of EGCG for 1 h prior to TNF-α stimulation significantly inhibited TNF-α-induced MCP-1 production in a concentration- dependent manner.

MCP-1 mRNA expression in HUVECs was assayed by RT-qPCR. As shown in Fig. 2, the level of MCP-1 mRNA in HUVECs was also markedly increased following exposure of the cells to 10 ng/ml TNF-α for 4 h. After the cells were preincubated with different concentrations of EGCG for 1 h, TNF-α-induced mRNA expression of MCP-1 was significantly down-regulated in a concentration-dependent manner. The study also demonstrated that EGCG alone did not affect the mRNA expression of MCP-1.

We performed a Cell counting kit-8 assay to assess the cell viability. The HUVECs were treated with 0, 10, 25 or 50μM EGCG for 24 h. As shown in Fig. 3, EGCG up to 50μM did not display any cellular toxicity against HUVECs. This finding suggested that the effects of EGCG were not due to cytotoxicity.

It is well known that NF-κB activation is required for the TNF-α-induced inflammatory cytokine production in HUVECs [7]. In order to determine whether the effect of EGCG on MCP-1 generation was due to suppression of NF-κB activation, we investigated the effects of EGCG on NF-κB activation in HUVECs. As shown in Fig. 4, HUVECs treated with TNF-α led to NF-κB activation, including IκB-α degradation and p65 phosphorylation. TNF-α-induced NF-κB activation was potently inhibits by EGCG treatment. We next evaluated the effects of EGCG on NF-κB-dependent transcriptional activity by transient transfection of HUVECs with a NF-κB-luciferase reporter plasmid. As shown in Fig. 5, TNF-α-induced NF-κB -dependent transcriptional activity was potently inhibited by EGCG in a dose-dependent manner.

As shown in Fig. 6 A and B, the protein and mRNA expression of 67LR in HUVECs were determined by western blot and RT-PCR analysis. It was next investigated whether the 67LR could mediate the inhibitory effects of EGCG on the TNF-α induction of MCP-1 expression. For this purpose, we treated HUVECs with neutralization antibody against 67LR (20 μg/ml) or control IgM for 1 h prior to TNF-α and/or EGCG treatment. As shown in Fig. 6 C, the blockage of 67LR attenuated the inhibitory effect of EGCG on the TNF-α induction of MCP-1 expression. Moreover, the inhibitory effect of EGCG on TNF-α-mediated NF-κB activation was counteracted by antibody against 67LR pre-treatment (Fig. 6 D). Our results suggest that the 67LR is involved in EGCG-mediated suppression of TNF-α-mediated MCP-1 generation.

We used monocyte adhesion assay to evaluate the physiological consequences of the inhibitory effect of EGCG on induction of MCP-1. As shown in Fig. 7, monocytes bound readily to TNF-α-activated HUVECs, whereas EGCG pretreatment for 1 h reduced adhesion of monocytes in a concentration dependent manner.

In the present study, we demonstrated that EGCG, a major green tea catechin, inhibited the expression MCP-1 induced by TNF-α in HUVECs. We found that EGCG reduced the MCP-1 mRNA and protein levels induced by TNF-α. EGCG treatment also inhibited monocytes adhesion to TNF-α-activated HUVECs in a concentration dependent manner. Our results provide the evidence for the potential development of EGCG in the prevention of atherosclerosis.

NF-κB acts as a central mediator for the regulation of genes important in immune and inflammatory responses [21]. To understand the potential mechanisms for EGCG attenuation of MCP-1 production by TNF-α, we confirmed in the HUVECs that a predominant signal transduction pathway by which TNF-α induces MCP-1 secretion is through inhibition of NF-κB activation. Our results demonstrated that EGCG significantly suppressed TNF-α-induced IκB-α degradation. On the other hand, we also observed that EGCG inhibited TNF-α-induced phosphorylation of NF-κB p65. Previous study has shown that EGCG significantly inhibits NF-κB transcriptional activity and nuclear translocation in RAW 264.7 macrophages [22]. Also it has been reported that EGCG can regulate the production of inflammatory cytokines via inhibiting the activation of NF-κB pathway in human mast cell line [23]. In addition, EGCG was suggested to have the ability to block NF-κB activation in intestinal epithelial cell line IEC-6 [24], respiratory epithelial cells [25], human endothelial ECV304 cells [26], and in hCMECs [16]. These are compatible with our data suggesting one mechanism of action for EGCG on endothelial cell function is the inhibition of the NF-κB pathway following TNF-α stimulation.

In our study, the inhibitory actions of EGCG were mediated through 67LR, suggesting that 67LR has a pivotal role as a cell-surface receptor that mediates the inhibitory action of EGCG in TNF-α-mediated MCP-1 generation in HUVECs. However, the expression of other inflammatory cytokines still warrants further investigation.

Green tea consumption is now considered to be an inexpensive, readily applicable, and accessible approach to CVD control and management [4]. In our previous study, we estimated that the risk of CAD decreased by 10% with each increase in green tea consumption of 1 cup/d [2]. Green tea catechins have been associated with a wide spectrum of beneficial effects on CAD; they have been found to inhibit vascular inflammation, oxidation, thrombogenesis, and atherogenesis and to favorably modulate the vascular reactivity and plasma lipid profile [2,4]. However, the mechanism of the chemopreventive action of most catechins, including EGCG, is still unknown. Our data suggested that EGCG down regulates MCP-1 expression in activated HUVECs; this may be a putative mechanism for the anti-atherosclerotic effects of green tea consumption.

In our study, the concentrations of EGCG (10-50μM) are generally within the range used in most published in vitro studies [27,28,29]. Nevertheless, the relatively high concentrations of EGCG obtained in cell culture studies have been considered to far exceed the levels found in human plasma after green tea consumption. The pharmacokinetic studies in humans demonstrated that the peak plasma concentration after single oral dose administration of EGCG is less than 1.0 μM [30,31]. Therefore, the rather poor bioavailability of EGCG needs to be considered when we extrapolate the activities observed with high EGCG doses in cell culture conditions to situations in vivo. However, efforts in improving the bioavailability of EGCG with promising approaches are being undertaken; recently reported attempts such as encapsulation of EGCG, the semisynthesis O-acyl derivatives of EGCG, the transdermal delivery of EGCG are worthy of further exploration to enhance the EGCG concentrations in the plasma [32].

Taken together, our results suggest that EGCG, a major polyphenol of green tea, could suppress TNF-α-induced MCP-1 mRNA and protein expression via inhibition of NF-κB in HUVECs. In addition, the present data demonstrate that the 67LR is involved in EGCG-mediated suppression of TNF-α-mediated MCP-1 generation. Our result shows that EGCG plays a protective role against endothelial dysfunction induced by TNF-α and may help us to better understand the beneficial effects of the green tea polyphenol proved in previous epidemiologic studies.

This work was supported by grants from the National Natural Science Foundation of China (No. 81270255 to L-SW and No. 30971254 to Z-JY), the Project Funded by the Science and Technological Innovation Group of Jiangsu Higher Education Institution “Qing-Lan Project” (JX2161015030) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).