[Protective effect of FAK inhibitor PF-562271 against human umbilical vein endothelial cell injury induced by aging platelets]

Abstract

Objective: To investigate the protective effect of PF-562271, a FAK inhibitor, against aging platelet-induced injury in human umbilical vein endothelial cells (HUVECs).

Methods: Cultured HUVECs were treated with vehicle, lipopolysaccharide (LPS), LPS+aging platelets, or LPS+aging platelets+PF-562271. The changes in protein expressions of FAK, pFAK and PECAM-1 in the treated cells were detected using Western blotting and immunofluorescence assay, and the level of reactive oxygen species (ROS) was detected with flow cytometry. The changes of barrier function of the cells were assessed with cell permeability test and transendothelial cell resistance test. RT-qPCR was used to analyze mRNA expressions of inflammatory factors, and pro-inflammatory cytokine levels in the culture supernatants was determined with enzyme-linked immunosorbent assay. Immunofluorescence assay was used to examine the effect of the ROS inhibitor vitamin C on PECAM-1 expression in the cells with different treatments.

Results: Treatment of HUVECs with LPS and aging platelets significantly increased cellular protein expressions of FAK, pFAK and PECAM-1, which were effectively lowered by addition of PF-562271 (P < 0.05). LPS and aged platelets obviously enhanced ROS production in the cells, which was inhibited by the addition of PF-562271 (P < 0.001). PF-562271 significantly alleviated the damage of endothelial cell barrier function of the cells caused by LPS and aging platelets (P < 0.01). The expressions of TNF-α, IL-6 and IL-8 in HUVECs increased significantly after exposure to LPS and aging platelets, and were obviously lowered after treatment with PF-562271 (P < 0.05). Treatment with vitamin C significantly decreased the expression of PECAM-1 protein in the cells (P < 0.01).

Conclusion: The FAK inhibitor PF-562271 alleviates endothelial cell damage induced by LPS and aging platelets by lowering cellular oxidative stress levels and reducing inflammatory responses.

Keywords: FAK; PECAM-1; PF-562271; TRALI; aging platelets; human umbilical vein endothelial cell.

图 1

PF-562271抑制FAK、pFAK表达 PF-562271 inhibits FAK and pFAK expression in cultured HUVECs. A-C: Western blotting for detecting changes in FAK and pFAK protein expressions. D: RTqPCR for FAK mRNA expression. Data are presented as Mean ± SD (n=3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

图 2

免疫荧光检测FAK表达减少 Expression of FAK protein in the treated HUVECs detected by immunofluorescence staining (Original magnification: ×40). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

图 3

PF-562271对内皮细胞屏障功能和通透性的影响 Effect of PF-562271 on barrier function and permeability of HUVECs with different treatments. A, B: Changes in ROS levels. C: Changes in TEER levels. D: Changes in cell permeability. Data are presented as Mean±SD (n=3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

图 4

PF-562271抑制TNF-α、IL-6、IL-8的表达 PF-562271 inhibits the expression of TNF-α, IL-6 and IL-8. A-C: TNF-α, IL-6 and IL-8 mRNA levels detected using RT qPCR. D-F: TNF-α, IL-6 and IL-8 protein levels detected using ELISA. (Mean±SD, n=3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

图 5

PF-562271抑制PECAM-1表达 PF-562271 inhibits PECAM-1 expression in cultured HUVECs. A-B: Western blotting for PECAM-1 protein expression changes. C: RT-qPCR for PECAM-1 mRNA expression changes. Data are presented as Mean±SD (n=3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

图 6

免疫荧光检测PECAM-1表达降低 Immunofluorescence staining of PECAM-1 expression in HUVECs with different treatments (×40). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

图 7

加入维生素C后，免疫荧光检测PECAM-1蛋白表达降低 Vitamin C decreases PECAM-1 protein expression in HUVECs with different treatments detected by immunofluorescence staining (×40). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.