Effects of (-)-epigallocatechin gallate on RPE cell migration and adhesion

Abstract

Purpose: In diseases such as proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR), and age-related macular degeneration (AMD), retinal pigment epithelial (RPE) cells can initiate proliferation and migration and secrete extracellular matrix (ECM) proteins. (-)-Epigallocatechin gallate (EGCG)-a natural anti-oxidant flavonoid that is abundant in green tea-has been shown to suppress the migration and adhesion of many cell types, but its effects on RPE cell migration and adhesion were unknown. Several studies have shown that platelet-derived growth factor (PDGF) enhances proliferation and migration effects on RPE cells in PVR, and that fibronectin is a major ECM component of PVR tissue. Therefore, we investigated the inhibitory effects of EGCG on RPE cell migration induced by PDGF-BB, an isoform of PDGF, and adhesion by fibronectin.

Methods: The migration of RPE cells was detected by an electric cell-substrate impedance sensing (ECIS) migration assay and a Transwell migration assay. Cells were loaded with 2',7'-bis-(carboxyethyl)-5(6')-carboxyfluorescein acetoxymethyl ester (BCECF/AM), and their adhesion to fibronectin was examined. The interactions of EGCG with PDGF-BB were analyzed by a dot binding assay. Cytoskeletal reorganization was examined by immunofluorescence microscopy. The PDGF-BB-induced signaling pathways were detected by western blotting.

Results: In the present study, we find that EGCG can inhibit PDGF-BB-induced human RPE cell migration and, in a dose-dependent manner, RPE cell adhesion to fibronectin. Our analysis demonstrates that EGCG does not directly bind to PDGF-BB and the inhibition of EGCG against fibronectin-induced cytoskeletal reorganization is observed. Furthermore, EGCG is shown to suppress PDGF-BB-induced PDGF-beta receptors, downstream PI3K/Akt, and MAPK phosphorylation.

Conclusions: Our results provide the first evidence that EGCG is an effective inhibitor of RPE cell migration and adhesion to fibronectin and, therefore, may prevent epiretinal membrane formation.

Figure 1

Impedance measurements on retinal pigment epithelium (RPE) cells as a function of time. Cultured adult human retinal pigment epithelial (ARPE19) cells were plated in chambers containing gold electrodes, and impedance measurements were made at fixed time intervals for 24 h. The epigallocatechin gallate (EGCG) inhibited ARPE19 cell migration, even without the presence of platelet-derived growth factor (PDGF)-BB. For the cells treated with 20 ng/ml PDGF-BB, the impedance increased faster and was significantly higher at 24 h than the cells treated without 20 ng/ml PDGF-BB (p<0.05) or with 10 μM EGCG (p<0.05). The experiment was performed four times with similar results. Each value represents the mean of four replicates.

Figure 2

Effects of epigallocatechin gallate (EGCG) on retinal pigment epithelium (RPE) cell migration. A, B: Epigallocatechin gallate inhibits platelet-derived growth factor (PDGF)-BB-induced RPE cell migration. Transwell inserts were coated with fibronectin. Human RPE cells were seeded in the upper chamber in the presence of vehicle or EGCG. The inserts were assembled in the lower chamber, which was filled with serum-free ([−]PDGF-BB; A) or PDGF-BB-containing medium ([+]PDGF-BB; B) and preincubated with a vehicle or EGCG on the polycarbonate filter of the insert for 30 min. PBS, without PDGF-BB or EGCG, served as the control (A, left). Human RPE cells that migrated to the underside of filter membrane were photographed and counted in high-power field (HPF, magnification, 100×) under a phase-contrast light microscope. The scale bar represents 100 μm. The black spots are the pores of the Transwell membrane and the grayish fusiform cells are the ARPE cells. C: Quantitative analysis of migrated cells. Twenty HPFs were counted in each migration. All experiments were conducted in duplicate and similar results were obtained at least two to three times. Data are presented as percent of control (the first unfilled bar, PBS only) in cell counts. *p<0.05 significantly differs from PDGF-BB-stimulated cells (the first filled bar).

Figure 3

Epigallocatechin gallate (EGCG) inhibited human retinal pigment epithelium (RPE) cell adhesion to fibronectin in a dose-dependent manner. Suspended RPE cells were loaded with 2’,7’-bis-(carboxyethyl)-5(6’)-carboxyfluorescein acetoxymethyl ester (BCECF/AM) and pretreated with different concentrations of EGCG. The cells were added to 96-well plates precoated with fibronectin (15 μg/ml) and incubated for 1 h at 37 °C. Cell adhesion was then measured by a fluorescence plate reader. Eight plates were quantified for each assay. Results are expressed as fluorescence intensity and represented by mean±SEM. *p<0.05 significantly differs from platelet-derived growth factor (PDGF)-BB-stimulated cells (the fifth bar).

Figure 4

Epigallocatechin gallate (EGCG) cannot directly interact with platelet-derived growth factor (PDGF)–BB in dot binding assay. Human recombinant PDGF-BB, phosphate buffer saline (PBS), the indicated concentrations of EGCG, and lycopene (Lyc) were applied onto the nitrocellulose (NC) membrane. The membrane was incubated with PDGF-BB in PBS and then developed by probing with Ab directed against PDGF-BB. Epigallocatechin gallate cannot directly interact with PDGF-BB, but lycopene can. The results presented are representative of three independent experiments.

Figure 5

Inhibition of fibronectin-induced human retinal pigment epithelial (RPE) cell cytoskeletal reorganization by Epigallocatechin gallate (EGCG). Suspended RPE cells pretreated with phosphate buffer saline (PBS, control) or 10 μM EGCG for 1 h were seeded and allowed to adhere on collagen-precoated glass coverslips for an additional 1 h. After fixation, permeabilization, and blocking with 3% BSA (BSA), cells on coverslips were incubated with fluorescein isothiocyanate (FITC)-phalloidin. Mounted cells were analyzed and photographed under a microscope. Actin formed in adherent cells (A), but there was a modification of cytoskeletal reorganization in EGCG-treated cells (B).

Figure 6

Effects of epigallocatechin gallate (EGCG) on PDGFR-β, PI3K, and MAPKs phosphorylation in human retinal pigment epithelium (RPE) cells. Platelet-derived growth factor-BB (10 ng/ml) was preincubated with a vehicle or 1, 3, and 10 μM of EGCG for 30 min and then added to human RPE cells. Western blotting was used to analyze PDGFR-β, PI3K, and MAPKs phosphorylation. All concentrations of EGCG at or above 3 μM significantly inhibited PDGF-BB-induced PI3K/Akt, ERK1/2, and p38 phosphorylation in human RPE cells in a concentration-dependent manner. However, there was no effect of PDGF-BB and EGCG on c-Jun N-terminal kinases (JNK) phosphorylation. The results presented are representative of four independent experiments.