The Reverse Effect Of EGCG On Ang â…¡-induced Cytoskeleton Reorganization In Endothelial Cells

Background and objectiveEpigallocatechin-3-gallate (EGCG) has protective effects on endothelial cells. Angiotensinâ…¡(Angâ…¡) is highly implicated in endothelial barrier dysfunction. Angâ…¡may cause endothelial cells damage by activating several signaling pathways. Changes in F-actin cytoskeleton induced by exoteric stimuli are closely associated with endothelial barrier dysfunction. Recently, a number of specific signaling molecules such as p38 MAPK,JNK1/2,and ERK1/2 are identified to modulate cytoskeleton reorganization. But how these signaling pathways orchestrate to regulate cytoskeleton reorganization and how they directly contribute to endothelial barrier dysfunction, induced by different exoteric stimuli in endothelial cells, are not fully illustrated.Therefore, we focus on the molecular mechanisms of Angâ…¡to modulate F-actin reorganization which leads to endothelial barrier dysfunction, and the reverse effects of EGCG.Methods and results1. To investigate the molecular mechanism of Angâ…¡to regulate F-actin reorganization in HUVECs.(1) F-actin was detected using Rhodamine-Phalloidin staining in HUVECs treated with Angâ…¡. We found that Angâ…¡induced F-actin reorganization. Observation by fluorescence microscopy showed that F-actin in normal cells concentrated along the cell membrane and well distributed. After treatment with Angâ…¡, most of the peripheral fibers disappeared, dense stress fibers were observed in the cytoplasm and paracellular gaps formed in a dose-and time-dependent manner.(2) Changes in the phosphorylated level of p38 MAPK, JNK1/2, and ERK1/2 were tested by Western blot analysis. It was showed that Angâ…¡promoted an increase in p38 MAPK and JNK1/2 phosphorylation, except ERK1/2. And the phosphorylated level of HSP27, the actin bonding protein, was also increased.(3) The MAPK inhibitors were investigated ahead of Angâ…¡treatment. The protein levels were determined by Western blot; F-actin was examined by rhodamine-phalloidin staining. Results showed that SB203580 significantly inhibited Angâ…¡-stimulated phosphorylation of p38 MAPK and HSP27 and blunted formation of stress fibers and paracellular gaps. Although JNK1/2 inhibitor SP600125 blocked JNK1/2 phosphorylation, it did not attenuated phosphorylation of HSP27 and formation of stress fibers.2. To study molecular mechanisms underlying the effect of EGCG on Angâ…¡-induced actin response in endothelial cells.Cells were pretreated with EGCG before stimulating with Angâ…¡. Western blot tested the phosphorylated levels of proteins, rhodamine-phalloidine staining examined the formation of actin stress fibers. EGCG inhibited Angâ…¡-induced phosphorylation of p38 MAPK and HSP27 and attenuated formation of stress fibers and paracellular gaps in a dose-dependent manner in HUVECs.ConclusionsAng II induces F-actin rearrangement in HUVECs, referring to disruption of actin filaments and an increased density of stress fibers, through activation of the p38 MAPK/HSP27 pathway. And EGCG attenuates these actions via inhibition of the p38 MAPK/HSP27 pathway. It may provide a novel insight into the performance of green tea polyphenol and suggest that EGCG be an effective protectant against endothelial barrier dysfunction and injury.