PEDF Protects Endothelial Barrier Integrity during Acute Myocardial Infarction via 67LR

Abstract

Maintaining the integrity and protecting the stability of tight junctions in endothelial cells is a potential therapeutic strategy against myocardial ischaemia. Laminin receptors (67LR) are highly expressed on endothelial cell membranes and are associated with endothelial barrier function. Herein, we sought to demonstrate the direct effects of pigment epithelial-derived factor (PEDF) on tight junctions between endothelial cells via 67LR during acute myocardial infarction (AMI) and elucidate its underlying mechanisms. We detected that PEDF directly increased the level of the tight junction protein zonula occludens protein 1 (ZO-1) after overexpression in vitro and in vivo using Western blotting. Evans Blue/TTC staining showed that PEDF significantly reduced the size of the infarcted myocardium. Immunofluorescence and the transwell cellular experiments suggested that PEDF significantly upregulated PI3K-AKT permeability and the distribution of ZO-1 between endothelial cells under OGD conditions. Interestingly, PEDF significantly upregulated the phosphorylation levels of PI3K-AKT-mTOR under oxygen and glucose deprivation conditions but had no significant effects on the total protein expression. The protective effect of PEDF on ZO-1 was significantly inhibited following the inhibition of PI3K-AKT-mTOR. The activation of phosphorylation of PI3K-AKT-mTOR by PEDF was blocked after silencing 67LR, as were the protective effects of PEDF on ZO-1. Therefore, we have reason to believe that PEDF increased ZO-1 expression through the 67LR-dependent PI3K-AKT-mTOR signaling pathway, thus maintaining tight junction stability and protecting cardiac function.

Keywords: 67LR; AMI; PEDF; ZO-1; endothelial cells.

Figure 1

PEDF reduces myocardial infarct size and vascular leakage in AMI rats. (A) Representative images of the Evans Blue/TTC staining in myocardial tissues. (B) Quantification of myocardial infarct size. (C) Quantification of serum cTn-I concentration in rats. (D,E) Western blotting to detect the effect of PEDF on the expression of ZO-1. (F,G) Immunofluorescence to observe the effect of PEDF on vascular leakage; 15–20 images from each group were used for fluorescence intensity analysis. White arrows point to leakages; Scale Bar = 100 μm. Data are expressed as mean ± SD; n = 4; NS, no significant difference; * p < 0.05; ** p < 0.01.

Figure 2

The effect of PEDF on the permeability of HUVECs during OGD. (A,B) Western blotting to detect the effect of PEDF on the expression of ZO-1 in vivo. (C) Immunofluorescence to observe the effect of PEDF on the distribution of ZO-1 in vivo. Scale Bar = 200 μm. (D,E) Western blotting to detect the effect of PEDF on the expression of ZO-1 in vitro. (F) Immunofluorescence to observe the effect of PEDF on the distribution of ZO-1 in vitro. Scale Bar = 200 μm (G,H) Quantification of the effect of PEDF on endothelial cell permeability. Data are expressed as mean ± SD; n = 4; NS, no significant difference; * p < 0.05; ** p < 0.01.

Figure 3

PEDF regulates the PI3K-AKT-mTOR pathway in endothelial cells during OGD. (A) Western blot to detect the effect of PEDF on phospho-PI3K, phospho-AKT and phospho-mTOR expressions. (B–E) Quantification of related protein expressions. Data are expressed as mean ± SD; n = 4; NS, no significant difference; * p < 0.05; ** p < 0.01.

Figure 4

PEDF maintains endothelial tight junction stability through 67LR. (A–C) Transfection with both 67LR and PEDFR viruses was successful. (D,E) Western blotting to detect the effect of PEDF on the expression of ZO-1 in vitro; (F) Immunofluorescence to observe the effect of PEDF on the distribution of ZO-1 in vitro. Scale Bar = 200 μm. Data are expressed as mean ± SD; n = 6; NS, no significant difference; * p < 0.05; *** p < 0.001.

Figure 5

PEDF regulates the PI3K-AKT-mTOR pathway and ZO-1 expression through 67LR. (A) Western blotting to detect the effect of PEDF on phospho-PI3K, phospho-AKT, phospho-mTOR, and ZO-1 expressions. (B–E) Quantification of the related protein expressions. Data are expressed as mean ± SD; n = 4; NS, no significant difference; * p < 0.05.

Figure 6

Schematic diagram demonstrating that PEDF maintains the stability of TJs by activating the PI3K-AKT-mTOR pathway via the 67LR receptor under OGD conditions.