Role of FXR in regulating PAI-1 expression and its impact on plasma fibrinolysis and DVT. (A-I) MPHs derived from WT or Fxr-null mice (FXRKO): (A) western blot analysis confirmed the knockout of FXR (N = 4); (B) Nr0b2 mRNA (N = 4); (C) Abcb11 mRNA (N = 4); (D) PAI-1 protein (N = 4); (E) PAI-1 concentration in medium (N = 4); (F-G) PAI-1 protein from TCA-treated MPH (N = 4); (H-I) PAI-1 protein from CDCA-treated MPHs (N = 4). (J) Serially truncated PAI-1 promoter constructs were cloned and inserted into pGL3-luciferase reporter plasmids and transfected into AML12 and HEK293 cells. Four hours after transfection, the cells were treated with CDCA (25 μM) for 24 hours and the relative luciferase activities were determined 72 hours after CDCA treatment. (K) A Ch-IP assay demonstrated the direct binding of FXR to the PAI-1 promoter in AML12 cells. (L) RT-qPCR of the Ch-IP products validated the binding capacity of FXR to the PAI-1 promoter. (M-O) WT and Fxr-null mice were randomly selected for DVT modeling or other assays: (M) tail bleeding time (N = 9); (N-O) thrombus weight and length (N = 5). (P) Representative curves for plasma fibrin formation and lysis tests and parameters of the curves for clot lysis time (N = 4). (Q-S) Fxr-null mice were injected with AAV8-TBG-Zsgreen or AAV8-TBG-shSerpine1: (Q) PAI-1 levels (N = 4); (R-S) thrombus length and weight (N = 6). WT mice were injected with AAV8-TBG-Zsgreen, and Fxr-null mice were injected with AAV8-TBG-Zsgreen or AAV8-TBG-shSerpine1: (T) representative curves for plasmin generation assay and parameters of the curves for EPP. (U-V) Fxrfl/fl mice were injected with AAV8-TBG-Cre or AAV8-TBG-Zsgreen: (U) plasma PAI-1 levels (N = 4); (V) thrombus weight and length (N = 5). The data are represented as the mean ± SEM, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 vs WT, Fxr-null and Fxrfl/fl mice with AAV8-TBG-Zsgreen or Veh group. ##P < .01 vs WT& FXR agonist (TCA or CDCA) group. BT, bleeding time; EPP, endogenous plasmin potential; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LUC, luciferase; NC, normal control; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Role of FXR in regulating PAI-1 expression and its impact on plasma fibrinolysis and DVT. (A-I) MPHs derived from WT or Fxr-null mice (FXRKO): (A) western blot analysis confirmed the knockout of FXR (N = 4); (B) Nr0b2 mRNA (N = 4); (C) Abcb11 mRNA (N = 4); (D) PAI-1 protein (N = 4); (E) PAI-1 concentration in medium (N = 4); (F-G) PAI-1 protein from TCA-treated MPH (N = 4); (H-I) PAI-1 protein from CDCA-treated MPHs (N = 4). (J) Serially truncated PAI-1 promoter constructs were cloned and inserted into pGL3-luciferase reporter plasmids and transfected into AML12 and HEK293 cells. Four hours after transfection, the cells were treated with CDCA (25 μM) for 24 hours and the relative luciferase activities were determined 72 hours after CDCA treatment. (K) A Ch-IP assay demonstrated the direct binding of FXR to the PAI-1 promoter in AML12 cells. (L) RT-qPCR of the Ch-IP products validated the binding capacity of FXR to the PAI-1 promoter. (M-O) WT and Fxr-null mice were randomly selected for DVT modeling or other assays: (M) tail bleeding time (N = 9); (N-O) thrombus weight and length (N = 5). (P) Representative curves for plasma fibrin formation and lysis tests and parameters of the curves for clot lysis time (N = 4). (Q-S) Fxr-null mice were injected with AAV8-TBG-Zsgreen or AAV8-TBG-shSerpine1: (Q) PAI-1 levels (N = 4); (R-S) thrombus length and weight (N = 6). WT mice were injected with AAV8-TBG-Zsgreen, and Fxr-null mice were injected with AAV8-TBG-Zsgreen or AAV8-TBG-shSerpine1: (T) representative curves for plasmin generation assay and parameters of the curves for EPP. (U-V) Fxrfl/fl mice were injected with AAV8-TBG-Cre or AAV8-TBG-Zsgreen: (U) plasma PAI-1 levels (N = 4); (V) thrombus weight and length (N = 5). The data are represented as the mean ± SEM, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 vs WT, Fxr-null and Fxrfl/fl mice with AAV8-TBG-Zsgreen or Veh group. ##P < .01 vs WT& FXR agonist (TCA or CDCA) group. BT, bleeding time; EPP, endogenous plasmin potential; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LUC, luciferase; NC, normal control; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

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