Intimal hyperplasia leading to restenosis is the major process that limits the success of cardiovascular intervention. Neointimal hyperplasia in response to arterial injury is a complex process, classically believed to be the consequence of vascular smooth muscle cell (VSMC) proliferation and migration, and the synthesis of extracellular matrix [1
]. The neointimal lesion induced by vascular injury evolves as a result of the synergistic interplay between the endothelial cell (EC) layer, platelets and VSMCs [2
]. Direct injury to vascular cells is an important stimulus for the production of autocrine-paracrine growth factors, such as PDGF-AA, TGF-β1, basic fibroblast growth factor and insulin-like growth factor I, that promote the proliferation of VSMCs, whereas blood-borne factors (e.g., PDGF-BB and thrombin) appear to be more important in promoting the migration of VSMCs into the subintimal space [3
]. There have been reported various therapeutic methods including chemotherapy, brachytherapy and gene therapy in order to suppress intimal hyperplasia [6
]. However, some treatments were effective in in vitro
or animal studies, whereas other studies have found little or no benefit in clinical applications [9
Polyphenols with strong antioxidant properties, derived from diverse dietary foods or beverages such as tea, grape, turmeric, etc
, have shown cancer chemopreventive and chemotherapeutic effects in many cell culture systems and animal tumor bioassays [10
]. Among various nutraceutical ingredients, epigallocatechin-3-gallate (EGCG), the most abundant and most active catechin derivative in green tea, predominantly accounts for the biological effects of green tea [10
]. Epidemiological and experimental studies have established a positive correlation between the consumption of green tea and protection against atherosclerosis and cardiovascular diseases [13
]. The underlying mechanisms of action involve vascular and myocardial effects of tea constituents [14
]. Mechanisms that have been suggested as being involved in the antiatherosclerotic effects of green tea consumption primarily entail antioxidative, anti-inflammatory, antiproliferative and antithrombotic properties, as well as beneficial effects on endothelial function [13
]. Many in vitro
and in vivo
studies have shown that green tea polyphenolic compounds (GTPCs) has potent antioxidative, radical-scavenging metal ions-chelating, and redox-sensitive transcription factors-inhibiting properties, which may partially account for their antiatherogenic effects [15
In the present study, we investigated the effect of EGCG on the cellular behaviors of vascular cells, ECs and SMCs although they were originated from different species, i.e. human and rat, respectively. Our data exhibit that the proliferation of both cells is dose-dependently decreased in response to increasing concentrations of EGCG, while the migration responses to EGCG are completely different between VECs and VSMCs. This differential effect of EGCG may be exploited to develop strategies for the prevention of neointima formation and subsequent restenosis by EGCG.
3.1. Cell Cultures
Animal care followed the criteria of the Animal Care Committee of Yonsei University College of Medicine for the care and use of laboratory animals in research. All experiments related to surgical procedures and treatments were performed in accordance with the guidelines of the Animal Experiment Committee of Yonsei University College of Medicine. VSMCs were isolated by limited enzymatic digestion from the tunica media of inferior vena cava of male young adult (9 ~ 10 wk old) Sprague-Dawley rats (280 ~ 300 g in weight, Samtako Inc., Gyeonggi-do, Korea) as previously reported [32
]. The primarily cultured VSMCs were maintained in a complete RPMI 1640 medium containing 11.1 mM D-glucose (Sigma–Aldrich Co., St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS, Sigma–Aldrich) and 1% antibiotic antimycotic solution (including 10,000 units penicillin, 10 mg streptomycin and 25 μg amphotericin B per ml, Sigma–Aldrich) at 37 °C and 5% CO2
in a humid environment. Studies were performed with the cells between third to fifth passage.
VECs were purchased from Lonza (human umbilical vein ECs, Walkersville, MD, USA) and cultured in endothelial cell basal medium-2 containing 11.1 mM D-glucose (Lonza) supplemented with 2% FBS (Lonza), EC growth supplements (Lonza; hEGF, hydrocortisone, hEGF, VEGF, hFGFB, R3-IGF-1 and ascorbic acid) and 1% antibiotic antimycotic solution (Sigma–Aldrich) at 37 °C in a humidified atmosphere of 5% CO2
in air as previously reported [34
]. Studies were performed with the cells between passages 3 to 5.
3.2. Morphological and Immunocytochemical Analyses of Primarily Cultured VSMCs
On day 5 after seeding the minced smooth muscle tissue explant onto a culture flask and on day 4 after the forth passage, the morphologies of the primarily cultured cells were respectively observed using a phase-contrast microscope (TE 300, Nikon, Osaka, Japan). Prior to seeding VSMCs into well plates, the cells at the fifth passage were characterized by a standard immunostaining method where mouse monoclonal anti-α-SMA antibody conjugated with FITC isomer I (Sigma–Aldrich) and PI (Sigma–Aldrich) for nucleus counterstaining were combined with fluorescence microscopy (Biozero – 8000, Keyence, Osaka, Japan) as previously described [32
3.3. Cytotoxicity of EGCG to VECs and VSMCs
The number of viable cells was quantified indirectly using a highly water soluble tetrazolium salt [WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, mono-sodium salt] (Dojindo Lab., Kumamoto, Japan), reduced to formazan dye by mitochondrial dehydrogenases. Thus, WST-8 assay was used to estimate the cytotoxicity of EGCG to VECs and VSMCs. Each cell culture with 2 × 104 cells/well as an initial seeding number was treated with increasing concentrations of EGCG and then incubated with WST-8 for the last 4 hr of the culture period (24 hr) at 37 °C in the dark. In order to avoid a direct reaction between EGCG and WST-8, the culture was thoroughly washed followed by the medium exchange before adding WST-8. Parallel sets of wells containing freshly cultured, non-treated cells were regarded as the controls. Absorbance was determined at 450 nm using an ELISA reader (Spectra Max 340, Molecular Device Inc., CA, USA). The relative cell viability (% of control) was expressed as the percentage of the optical densities in EGCG-treated wells to the optical density in a non-treated well. From the cytotoxicity profile, IC50 value (μM) was defined as the concentration of EGCG in culture media which decreases cell viability down to 50%.
3.4. EGCG Treatments
EGCG (Teavigo™, ≥98% purity) was purchased from DSM Nutritional Products (Basel, Switzerland). In order to examine the effects of EGCG on the proliferation of VECs and VSMCs, both cells were seeded into well plates and then incubated in the presence of increasing concentrations (0 ~ 400 μM) of EGCG in their respective complete medium for 24 hr. Upon determining the differential effects of EGCG on the migration of VECs vs. VSMCs, each cell was treated with 200 μM EGCG in each complete medium for up to 36 hr.
3.5. Cell Proliferation Assay
Cell proliferation was found to be directly proportional to the metabolic reaction products obtained in WST-8. As describe above, the proliferation of VECs and VSMCs exposed to increasing concentrations of EGCG were determined by WST-8 assay. Both cells were treated with EGCG and then incubated with WST-8 for 4 hr followed by measuring absorbance at 450 nm.
3.6. Cell Migration Assay
As described in our previous studies [35
], in vitro
migration assays were performed. In brief, VECs or VSMCs (1 × 105
cells/mL) were seeded in 4-well chambered cover-glass slide and grown to confluence overnight. Mono-layers were scraped (denuded) using a 1 mL plastic micropipette tip, and 200 μM EGCG was treated to the confluent cell layers. EGCG-treated or non-treated cells were incubated in a self-designed CO2
mini-incubator placed on the stage of an inverted system microscope (IX70, Olympus Optical Co., Osaka, Japan) and then visualized for cells migrating into denuded space by a CCD camera (Olympus Optical Co.) attached to the microscope. Migration of cells into denuded areas was monitored for up to 36 hr. The average area covered by migrated cells and the migration speed of an individual cell were calculated by using image processing software (MATLAB V5.3, Mathwork Inc., Natic, MA, USA).
3.7. Statistical Analysis
All variables were tested in three independent cultures for each experiment, and each experiment was repeated twice (n = 6). The results are reported as a mean ± standard deviation (SD) compared with the non-treated controls. A one-way analysis of variance (ANOVA), which was followed by a Tukey HSD test for the multiple comparisons, was used to detect the effects of EGCG on VECs and VSMCs. A p value < 0.05 was considered statistically significant.
In conclusion, both vascular cells were significantly affected by EGCG of which the higher concentrations (over 200 μM) inhibited the more cell proliferation. However, EGCG exerted differential migration-inhibitory activity in VECs vs. VSMCs. EGCG-mediated inhibition of migration was found to occur at much higher dose of EGCG in VECs as compared to VSMCs. Although this kind of peculiar response of VECs to EGCG could not be obviously explained at present, it would be suggested that EGCG might be exploited as an efficient drug for a stent due to its selective activity, completely suppressing the proliferation and migration of VSMCs, but not adversely affecting VECs migration in blood vessels.