Epigallocatechin-3-Gallate Attenuates Myocardial Dysfunction via Inhibition of Endothelial-to-Mesenchymal Transition

Figures

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  Figure 1. EndMT induction by TGF-β2 and IL-1β and inhibition by EGCG in HUVECs. (A) Microscope images of HUVECs in the control and TGF-β2 and IL-1β-treated groups (100× magnification). (B) Structural formula of EGCG. (C) CCK-8 cell viability assay of HUVECs treated with EGCG. (D,E) Effect of EGCG on endothelial and mesenchymal cell marker expression levels in HUVECs with TGF-β2 and IL-1β co-stimulation compared with the control group. (F) Fluorescence microscopy image of FL-LDL uptake in HUVECs (40× magnification). (G) Dextran permeability assay HUVEC monolayer. ### p < 0.001 vs. HUVECs without EGCG and TGF-β2 and IL-1β treatment; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TGF-β2- and IL-1β-induced HUVECs without EGCG treatment. Data were fold-changed for control group (n = 3); N.S., not significant.

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  Figure 2. EGCG-induced inhibition of cell migration ability caused by TGF-β2 and IL-1β treatment. (A) Microscope images of scratched area observed at 0 h and 24 h after making scratch (10× magnification) and (B) percentage of recovered area after 24 h. (C,D) Effect of EGCG on the expression level of RhoA and phosphorylation level of CDC42 and cofilin in HUVECs co-stimulated with TGF-β2 and IL-1β compared with control group. ## p < 0.01, ### p < 0.001 vs. HUVECs without EGCG and TGF-β2 and IL-1β treatment; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TGF-β2- and IL-1β-induced HUVECs without EGCG treatment. Data were fold-changed for control group (n = 3).

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  Figure 3. Antioxidant and anti-inflammatory effects of EGCG. (A) Fluorescence microscopy image of intracellular ROS produced after TGF-β2 and IL-1β co-stimulation with or without EGCG treatment (40× magnification). (B) ROS quantitative analysis. (C) Mitochondrial membrane potential measured via TMRE assay (200× magnification). (D) TMRE fluorescence intensity. (E,F) Effect of EGCG on phosphorylation level of NF-κB, IκBα, and SMAD2/3 in TGF-β2 and IL-1β co-stimulated HUVECs compared with control group. ### p < 0.001 vs. HUVECs without EGCG and TGF-β2 and IL-1β treatment; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TGF-β2- and IL-1β-induced HUVECs without EGCG treatment. Data were fold-changed for control group (n = 3).

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  Figure 4. Regulation of EndMT in EGCG-treated MI model. (A) Timeline of the animal study. (B) Changes in EndMT and EndMT-related gene expression (Col1a, Fsp1, Snail, Tgfb, and vimentin) according to real-time PCR (n = 3). (C) Snail expression according to immunofluorescence [green: Snail (EndMT marker), blue: DAPI] (10× magnification). High-quality version: Supplementary Figure S1. (D,E) Immunoblot analysis of collagen I, collagen III, MMP-2, and MMP-9 with GAPDH as control (n = 3). # p < 0.05 and ### p < 0.001 vs. Normal; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. MI. Data are expressed as X-fold induction compared to normal control. All values are mean ± standard deviation. Statistical significance was assessed by one-way ANOVA.

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  Figure 5. Cardioprotective effects of EGCG on heart tissue after ischemia. (A) Infarct size according to TTC staining. Infarct size (%) was calculated as the ratio of the infarcted area (pale) to the risk area (deep-red). Distance between bars = 0.1 cm. p = 0.0007 (n = 3). (B) Apoptotic cell death according to TUNEL assay (TUNEL-positive cells, pink; 7-AAD, blue) performed 1 week after MI (40× magnification). * p = 0.0004 (MI: n = 3; MI + EGCG: n = 4). High-quality version: Supplementary Figure S2. (C) Fibrotic tissue area measured using Masson’s trichrome of cardiac sections 1 week after establishing MI (collagen fibers, light blue; muscle fibers, red). Bar = 2 mm; p = 0.0013 (n = 3). (D) Capillary density according to immunofluorescence staining for CD31 1 week after MI (CD31-positive microvessels, red; DAPI, blue) (40× magnification). * p = 0.0035 (n = 5). High-quality version: Supplementary Figure S3. (E) Ejection fraction (n = 3). (F) Representative pressure–volume loop. (G) End-systolic pressure–volume relationship (ESPVR). (H) A schematic summary of the regulation of EndMT by treating EGCG in ischemic hearts. * p < 0.05 vs. MI group. Data are expressed as X-fold induction compared…