Ephrin B2 mediates high glucose induced endothelial-to-mesenchymal transition in human aortic endothelial cells

Abstract

Background: Previous study revealed that high glucose (HG) induced endothelial cell (EC) damage via endothelial-to-mesenchymal transition (EndMT). Recent studies suggested the role of Ephrin B2 in mediate ECs damage. However, the underlying mechanism remains unclear. The aim of the present study was to investigate whether Ephrin B2 mediates HG-induced EndMT in human aortic ECs (HAECs) and to determine the possible downstream signaling effector.

Methods: Primary HAECs were exposed to normal glucose (NG, 5.5 mM), HG (30 mM) and HG+Ephrin B2 small interfering RNA (siRNA), respectively. The pathological changes were investigated by light microscope and confocal microscopy. To study the effects of focal adhesion kinase (FAK) activation on Ephrin B2 in HAECs, cells were incubated with FAK siRNA in HG group. The expression of EndMT-related markers (CD31 and FSP1), Ephrin B2 and FAK were detected by qRT-PCR and western blot.

Results: The results showed that HG significantly inhibited the expression of CD31 and increased FSP1 compared with NG group. Moreover, Ephrin B2 was increased after HG incubation. Ephrin B2 siRNA attenuated HG-induced expression of EndMT-related markers. Furthermore, HG increased the expression of FAK and phosphorylated FAK (pho-FAK) in HAECs. In contrast, blocking Ephrin B2 could partially attenuate HG-induced FAK activation. And FAK siRNA further inhibited the EndMT-related markers in HAECs treated with HG.

Conclusions: HG-induced EndMT in HAECs might be partially mediated by Ephrin B2 and the downstream FAK pathway.

Keywords: Ephrin B2; endothelial-to-mesenchymal transition (EndMT); focal adhesion kinase (FAK); high glucose (HG); human aortic endothelial cell (HAEC).

Figure 1

Exposure to high glucose induces EndMT in HAECs. (A) Bright field demonstrating the morphological changes in HAECs cultured with normal glucose (NG, 5.5 mM) and high glucose (HG, 30 mM) for 48 hours. Scale bars, 100 µm. (B) mRNA levels of CD31 and FSP1 were detected by qRT-PCR. (C) Protein levels of CD31 and FSP1 were detected via western blot. (D) Results of the quantitative protein level. (E) Representative immunofluorescence images demonstrating CD31 (red) staining in HAECs treated with NG or HG for 48 hours; the nuclei were stained with DAPI (blue). Scale bar, 20 µm. Data were exhibited as means ± SD. *, P<0.05 vs. NG. Experiments were repeated four times. HG, high glucose; NG, normal glucose; EndMT, endothelial-to-mesenchymal transition; HAECs, human aortic endothelial cells; FSP1, fibroblast-specific protein 1.

Figure 2

Effects of high glucose on Ephrin B2 mRNA and protein expression in HAECs. (A) HAECs were cultured with increased glucose (5.5, 15, 30 mM) for 48 hours. Mannitol (MN) was used as a control hyperosmolarity. mRNA levels of Ephrin B2 were detected by qRT-PCR. (B) Representative blots for Ephrin B2. (C) Graphic presentation. Data were exhibited as means ± SD. *, P<0.05 vs. NG or MN. **, P<0.01 vs. NG or MN. Experiments were repeated five times. HAECs, human aortic endothelial cells; NG, normal glucose.

Figure 3

Blocking Ephrin B2 inhibited high glucose induced EndMT in HAECs. (A) We performed gene-silencing experiments using transfection with siRNA specific for Ephrin B2. HAECs were incubated with Ephrin B2-siRNA in the presence of HG. The mRNA levels of Ephrin B2, CD31 and FSP1 were measured by qRT-PCR. (B) Representative blots for Ephrin B2, CD31 and FSP1. (C) Graphic presentation. Data were exhibited as means ± SD. *, P<0.05 vs. HG. Experiments were repeated three times. EndMT, endothelial-to-mesenchymal transition; HAECs, human aortic endothelial cells; HG, high glucose; FSP1, fibroblast-specific protein 1.

Figure 4

Ephrin B2 blockade suppressed the high glucose induced FAK pathway. (A) siRNA for Ephrin B2 were performed. Protein levels of FAK and phosphorylated FAK were detected by western blot. (B) Results of quantitative protein levels. were exhibited as means ± SD. *, P<0.05 vs. NG. ^#, P<0.05 vs. HG. Experiments were repeated four times. FAK, focal adhesion kinase; HG, high glucose; NG, normal glucose.

Figure 5

FAK siRNA inhibits the high glucose induced EndMT. (A) siRNA for FAK were performed. Protein levels of phosphorylated FAK, CD31 and FSP1 were detected by Western blot. (B) results of quantitative protein levels. were exhibited as means ± SD. *, P<0.05 vs. HG. Experiments were repeated three times. EndMT, endothelial-to-mesenchymal transition; FAK, focal adhesion kinase; HG, high glucose.

Figure 6

Schematic diagram of this study. Ephrin B2 expression was up-regulated after high glucose treatment. High glucose-induced Ephrin B2 signaling triggered EndMT and endothelial damage, which mediated by the downstream FAK pathway. EndMT, endothelial-to-mesenchymal transition; FAK, focal adhesion kinase.