Focal Adhesion Kinase Inhibitor Inhibits the Oxidative Damage Induced by Central Venous Catheter via Abolishing Focal Adhesion Kinase-Protein Kinase B Pathway Activation

Abstract

The vascular injury induced by central venous catheter (CVC) indwelling is the basis for the occurrence and development of CVC-related complications, such as phlebitis, venous thrombosis, and catheter-related infections. Focal adhesion kinase (FAK) and FAK-protein kinase B (AKT) signaling pathway are of great significance in tissue repair after trauma. Here, we investigated the role and mechanism of the FAK inhibitor (1,2,4,5-phenyltetramine tetrahydrochloride (Y15)) in oxidative damage caused by CVC. EA.hy926 cells were divided into the control group (normal control), CVCs+scratches group (the intercepted CVC segments coculturing with scratched EA.hy926 cells), and CVCs+scratches+Y15 group (Y15 was added to the cell culture supernatant with CVCs + scratches at a final concentration of 50 μmol·L^-1). New Zealand rabbits were randomly divided into the control group (normal control), CVC group (CVC was inserted through the rabbit's right jugular vein to the junction of the right atrium and superior vena cava), and CVC+Y15 group (CVC was immersed in a 50 μmol·L^-1 Y15 solutions before insertion). The levels of markers and proteins related to oxidative damage in cells, cell culture supernatant, serum, and external jugular vein were measured by commercial kits and western blot, respectively. We found that Y15 treatment significantly decreased ROS and MDA levels and increased cell viability, NO, and SOD levels in a time-dependent manner in rabbit serum and cell culture supernatant. In addition, Y15 effectively reduced the CVC-induced pathological changes of damaged vascular tissues. Y15 also downregulated the levels of p-FAK Tyr 397 and p-Akt Ser 473 in damaged external jugular vein and EA.hy926 cells. These findings suggest that Y15 alleviated CVC-induced oxidative damage to blood vessels by suppressing focal FAK-Akt pathway activation.

Figure 1

Histopathological changes of the right external jugular vein. HE staining was performed to evaluate the histopathological changes. Representative images are shown. (a) Control group (×10). (b) CVC group at week 2 (×10): vascular intima shedding (black arrow), subintimal hemorrhage (green arrow) and mucoid degeneration (yellow arrow). (c) CVC group at week 4 (×10): granulation tissue organization in venous lumen (black arrow) and inflammatory cell infiltration (yellow arrow). (d) CVC group at week 6 (×20): scar tissue formation under the intima (black arrow). (e) CVC+Y15 group at week 2 (×20: inflammatory cell infiltration (black arrow) and hyaline degeneration (yellow arrow). (f) CVC+Y15 group (×10) at week 4: vascular intima shedding (black arrow). (g) CVC+Y15 group at week 6 (×10). Scale bar: 200 μm.

Figure 2

Y15-impregnated CVCs decreased the degree of oxidative damage of external jugular veins. ROS (a), MDA (b), NO (c), and SOD (d) levels in vein tissue were determined using ROS, MDA, NO, and SOD test kits, respectively. Data are presented as the mean ± SD (n = 5 for each time point per group). p values (p < 0.05) were calculated by two-way ANOVA analysis with the Bonferroni multiple comparison test. ^∗∗∗p < 0.001.

Figure 3

Western blot was performed to determine expression of total FAK, Akt, p-FAK Tyr 397, and p-Akt Ser 473. (a, b) The expression of total FAK, p-FAK Tyr 397, total Akt, and p-Akt Ser 473 in the CVC group and CVC+Y15 group. (c, d) The expression levels of p-FAK Tyr 397 and p-Akt Ser 473 at 2-week, 4-week, and 6-week time points were compared. Data are presented as the mean ± SD (n = 5 for each time point per group). Statistical significance was set at p < 0.05 and was determined by two-way ANOVA analysis was performed using Bonferroni multiple comparison test. ^∗∗∗p < 0.001.

Figure 4

Effect of Y15 on the cell viability of damaged EA.hy926 cells. Cell viability was detected with MTT assay. Data are presented as the mean ± SD (n = 5 for each time point per group). Two-way ANOVA analysis was performed using the Bonferroni multiple comparison test to determine p values (p < 0.05). ^∗∗∗p < 0.001.

Figure 5

Treatment of damaged EA. hy926 cells with Y15 decreased ROS and MDA levels and increased concentration of SOD and NO. ROS (a), MDA (b), NO (c), and SOD (d) levels in cells were measured with ROS, MDA, SOD, and NO test kits. Data are presented as the mean ± SD (n = 5 for each time point per group). p values (p < 0.05) were calculated by two-way ANOVA analysis with the Bonferroni multiple comparison test. (a, b) ^∗∗∗p < 0.001.

Figure 6

Y15 decreased protein levels of p-FAK, Tyr 397, and p-Akt Ser 473. Western blot was performed to determine expression of total FAK, Akt, p-FAK Tyr 397, and p-Akt Ser 473 at 24 h, 48 h, and 72 h. (a, c) The expression of total FAK and p-FAK Tyr 397 in the CVCs+scratches group and CVCs+scratches+Y15 group. (b, d) The protein levels of total Akt and p-Akt Ser 473. ^∗∗∗p < 0.001.

Figure 7

Schematic figure of Y15 inhibiting oxidative damage via the FAK-Akt signaling pathway. In oxidative damage models established by coculturing CVC segments with scratched EA.hy926 cells or inserting and retaining CVC in the vein of rabbits, overproduction of ROS and activation of FAK-Akt signaling triggered the changes in levels of biomarkers of oxidative stress (NO, MDA, and SOD), which resulted in cell injury or vein injury. Y15 alleviated the oxidative damage through inhibition of FAK-Akt pathway activation.