FXII regulates the formation of deep vein thrombosis via the PI3K/AKT signaling pathway in mice

Abstract

Deep vein thrombosis (DVT) is a common peripheral vascular disease, which may result in pulmonary embolism and is accompanied by endothelial injury. However, the pathogenesis of DVT remains unclear. Coagulation factor XII (FXII), as an important coagulation factor, has been reported to be closely associated with thrombosis. However, the association between FXII protein and DVT formation is not yet fully understood. The present study examined the effects of FXII protein on DVT formation and aimed to reveal the underlying mechanism. In the present study, histological characterization of the femoral vein tissue was examined by hematoxylin and eosin staining. The damage to the femoral vein tissue was examined by TUNEL assay. Superoxide dismutase (SOD) and malondialdehyde (MDA) concentrations were examined using ELISA. Tumor necrosis factor (TNF)‑α, interleukin (IL)‑6, IL‑8 and phosphoinositide 3‑kinase (PI3K)/AKT signaling were determined by ELISA, immunohistochemical staining and western blot analysis. The results demonstrated that thrombosis, FXII protein, cell apoptosis and the SOD concentrations were decreased, while the MDA concentrations were increased in mice with DVT compared with the control or sham groups. TNF‑α, IL‑6, IL‑8 and PI3K/AKT signaling was also upregulated in the mice with DVT. Furthermore, the knockdown of FXII significantly upregulated the SOD concentrations and downregulated thrombosis and cell apoptosis, as well as the MDA concentrations in mice with DVT. The knockdown of FXII also significantly downregulated the protein expression of TNF‑α, IL‑6 and IL‑8, and the activation of PI3K/AKT signaling. Additionally, LY294002 pre‑treatment markedly downregulated thrombosis and cell apoptosis and the MDA content, whereas it upregulated the SOD concentrations in mice with DVT. LY294002 pre‑treatment also significantly downregulated the TNF‑α, IL‑6 and IL‑8 protein levels. Taken together, the present study demonstrates that FXII protein promotes DVT via the activation of PI3K/AKT signaling by inducing an inflammatory response. Targeting FXII protein may thus prove to be a potential approach for the treatment of DVT.

Keywords: deep vein thrombosis; coagulation factor XII; PI3K/AKT signaling; inflammatory response.

Figure 1

FXII protein expression in the femoral vein tissue of mice with DVT. (A) Thrombosis in the femoral vein tissue of different treatment groups (n=6 mice in each group) was examined by H&E staining at 48 h after surgery (black arrows indicate the femoral vein tissue; red arrows indicate thrombosis). (B) Cell apoptosis of the femoral vein tissue in mice was examined by TUNEL assay. (C and D) SOD and MDA concentrations in the femoral vein tissue of mice were examined using ELISA. (E) FXII protein expression was examined by immunohistochemical staining. (F) FXII protein expression was examined by western blot analysis. GAPDH was used as the internal control. (G) Western blot analysis quantification of FXII protein expression. ^*P<0.05 and ^**P<0.01; ns, not significant. FXII, coagulation factor XII; DVT, deep vein thrombosis; SOD, superoxide dismutase; MDA, malondialdehyde; H&E, hematoxylin and eosin.

Figure 2

Expression of TNF-α, IL-6 and IL-8 protein in the femoral vein tissue and plasma of mice with DVT. (A, C and E) The levels of TNF-α, IL-6 and IL-8 in the femoral vein tissue of different treatment groups (n=6 mice in each group) were detected using ELISA. (B, D and F) The levels of TNF-α, IL-6 and IL-8 in the plasma of mice in the different treatment groups (n=6 mice in each group) were detected using ELISA. ^**P<0.01, ^***P<0.001 and ^****P<0.0001; ns, not significant. DVT, deep vein thrombosis.

Figure 3

Activation of PI3K/AKT signaling in the femoral vein tissue of mice with DVT. (A and B) The levels of PI3K, p-PI3K, AKT and p-AKT in the femoral vein tissue of different treatment groups (n=6 mice in each group) were determined by western blot analysis and immunohistochemical staining. (C and D) Western blot analysis quantification of PI3K, p-PI3K, AKT and p-AKT levels. ^**P<0.01; ns, not significant. DVT, deep vein thrombosis; p, phosphorylated.

Figure 4

Effects of FXII protein on the thrombosis of femoral vein tissue of mice with DVT. FXII protein was knocked down by transfection with pAd-pG2.1-siRNA FXII (1×10⁸ pfu/mice). (A and B) Western blot analysis quantification of the expression level of FXII protein in the femoral vein tissue of mice. (C) Thrombosis in the femoral vein tissue of different treatment groups (n=6 mice in each group) was examined by H&E staining (black arrows indicate the femoral vein tissue; red arrows indicate thrombosis). (D) Cell apoptosis of the femoral vein tissue in mice was examined by TUNEL assay. (E and F) SOD and MDA concentrations in the femoral vein tissue of mice were examined by ELISA. ^**P<0.01; ns, not significant. FXII, coagulation factor XII; DVT, deep vein thrombosis; siRNA, small interfering RNA; pfu, plaque-forming units; H&E, hematoxylin and eosin.

Figure 5

Effects of FXII protein on TNF-α, IL-6 and IL-8 protein in the femoral vein tissue and plasma of mice with DVT. FXII protein was knocked down by transfection with pAd-pG2.1-siRNA FXII (1×10⁸ pfu/mice). (A, C and E) The levels of TNF-α, IL-6 and IL-8 in the femoral vein tissue of different treatment groups (n=6 mice in each group) were detected using ELISA. (B, D, and F) The levels of TNF-α, IL-6 and IL-8 in the plasma of mice in the different treatment groups (n=6 mice in each group) were detected using ELISA. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001; ns, not significant. FXII, coagulation factor XII; DVT, deep vein thrombosis; siRNA, small interfering RNA; pfu, plaque-forming units.

Figure 6

Effects of FXII protein on the activation of PI3K/AKT signaling in the femoral vein tissue of DVT mice. FXII protein was knocked down by transfection with pAd-pG2.1-siRNA FXII (11×10⁸ pfu/mice). (A and B) The levels of PI3K, p-PI3K, AKT and p-AKT in the femoral vein tissue of different treatment groups (n=6 mice in each group) were determined by western blotting and immunohistochemical staining. (C and D) Western blot analysis quantification of PI3K, p-PI3K, AKT and p-AKT levels. ^*P<0.05, ^**P<0.01 and ^****P<0.0001; ns, not significant. FXII, coagulation factor XII; DVT, deep vein thrombosis; siRNA, small interfering RNA; pfu, plaque-forming units; p, phosphorylated.

Figure 7

Effects of PI3K/AKT signaling on the thrombosis of femoral vein tissue of DVT. The activation of PI3K/AKT signaling was inhibited by pre-treatment with LY294002 (50 mg/kg). (A) Thrombosis in the femoral vein tissue of different treatment groups (n=6 mice in each group) was examined by H&E staining (black arrows indicate the femoral vein tissue; red arrows indicate thrombosis). (B) Cell apoptosis of the femoral vein tissue in mice was examined by TUNEL assay. (C and D) SOD and MDA concentrations in the femoral vein tissue of mice were examined by ELISA. ^*P<0.05 and ^**P<0.01. DVT, deep vein thrombosis; H&E, hematoxylin and eosin; SOD, superoxide dismutase; MDA, malondialdehyde.

Figure 8

Effects of PI3K/AKT signaling on TNF-α, IL-6 and IL-8 protein in the femoral vein tissue and plasma of DVT mice. The activation of PI3K/AKT signaling was inhibited by pretreatment with LY294002 (50 mg/kg). (A, C and E) The levels of TNF-α, IL-6 and IL-8 in the femoral vein tissue of different treatment groups (n=6 mice in each group) were detected by ELISA. (B, D, and F) The levels of TNF-α, IL-6 and IL-8 in the plasma of different treatment groups (n=6 mice in each group) were detected using ELISA. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001. DVT, deep vein thrombosis.