Fig 1: The increase in hepatocyte PAI-1 drives the increase in hepatocyte tPA in obesity.(A) Serpine1fl/fl mice were fed a high-fat diet for 4 months and then injected intravenously with either AAV8-TBG-Cre or control AAV8-TBG-LacZ. After 4 weeks, liver Plat mRNA and plasma tPA protein concentrations were measured. Horizontal lines in the dot-density plots indicate mean values. n = 11 mice/group. *P < 0.05; **P < 0.01, 2-tailed Student’s t test. (B) Human primary hepatocytes were treated for 16 hours with 100 μM palmitate (PALM) in BSA solution, or BSA solution control. The cells were assayed for PLAT and SERPINE1 mRNA, and the media were assayed for tPA and PAI-1 protein concentration. (C) Human primary hepatocytes were transfected with siSERPINE1 or scrambled control (Scr). After 24 hours, cells were treated with 100 μM palmitate for an additional 16 hours and then assayed for PLAT and SERPINE1 mRNA. In B and C, results are shown as mean ± SEM. n = 3 sets of cells/group. *P < 0.05; **P < 0.01, 2-tailed Student’s t test. (D) Liver specimens from the subjects described in Supplemental Table 1 were assayed for PAI‑1/GAPDH densitometric ratio by immunoblot, as shown in Supplemental Figure 3C, and for PLAT mRNA. These data were then subjected to correlation analysis, as described in the legend for Figure 2B.
Fig 2: PAI-1 induces CREB1 phosphorylation, which then induces tPA expression in hepatocytes.(A) Livers of lean and obese mice were assayed for p-CREB1 and total CREB1 by immunoblot (n = 3 mice/group). (B) Serpine1fl/fl mice were fed a high-fat diet for 4 months and then injected intravenously with either AAV8-TBG-Cre or control AAV8-TBG-LacZ. After 4 weeks, livers were assayed for p-CREB1 and total CREB1 by immunoblot, with densitometric quantification shown. Data are shown as mean ± SEM. *P < 0.05, 2-tailed Student’s t test. (C) Human primary hepatocytes were transfected with siSERPINE1 or scrambled control. After 24 hours, cells were treated with 100 μM palmitate for 16 hours, followed by assay of phosphorylated and total CREB1 by immunoblot, with densitometric quantification shown. Data are represented as mean ± SEM. *P < 0.05, 2-tailed Student’s t test. (D and E) Human primary hepatocytes were transfected with siCREB1 or scrambled control. After 24 hours, cells were treated for 8 hours with 1 μg rPAI-1/mL culture medium or vehicle control (Veh). Cells were then assayed for phosphorylated and total CREB1 by immunoblot and for PLAT mRNA by qPCR. n = 3 sets of cells/group. Data are represented as mean ± SEM. *P < 0.05, 1-way ANOVA followed by Tukey’s test. (F) Nuclear extracts from the livers of lean or obese mice were subjected to ChIP assay using anti-CREB1 or control IgG (Ctrl-IgG). The proximal promoter region containing the CREB1-binding sequence in the Plat gene was amplified by qPCR and normalized to the values obtained from input DNA. n = 3 mice/group. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01, 1-way ANOVA followed by Tukey’s test.
Fig 3: Decreased Rev-Erbα in the livers of obese mice and in palmitate-treated primary human hepatocytes elevates both PAI-1 and tPA expression.(A and B) Livers of lean and DIO mice were assayed for Rev-Erbα protein and β-actin loading control by immunoblot, with densitometric quantification shown, and for Nr1d1 mRNA. n = 5–6 mice/group. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01, 2-tailed Student’s t test. (C) Lean and obese mice were injected intravenously with AAV8-TBG-Nr1d1 to increase Rev-Erbα expression in hepatocytes or with AAV8-TBG-LacZ control, as indicated. After 4 weeks, liver Serpine1 and Plat mRNA levels were measured. Horizontal lines in dot-density plots indicate mean values. n = 10 mice/group. *P < 0.05; **P < 0.01, 1-way ANOVA followed by Tukey’s test. (D) Human primary hepatocytes were transfected with a plasmid expressing Nr1d1 to increase expression of Rev-Erbα; transfection with empty vector (Vec) served as the control. After 24 hours, cells were treated with 100 μM palmitate for 16 hours and then assayed for Rev-Erbα protein by immunoblot and for SERPINE1 and PLAT mRNA by quantitative PCR (qPCR). n = 3 sets of cells/group. Data are represented as mean ± SEM. *P < 0.05, 1-way ANOVA followed by Tukey’s test.
Fig 4: Hepatocyte tPA is increased in DIO mice, but a larger increase in hepatocyte PAI-1 causes a net impairment of fibrinolysis.(A–C) Chow-fed mice (Lean) or DIO mice were injected intravenously with AAV8-H1-shPlat (shPlat) to silence the tPA gene Plat in hepatocytes or with control AAV8-H1-Scr (Ctrl). After 4 weeks, the following parameters were measured: (A) liver Plat mRNA; (B) plasma tPA protein concentration and plasma tPA activity; (C) fibrinolytic activity measured by euglobulin clot-lysis assay; and (D) time to occlusive carotid arterial thrombosis induced by rose bengal/laser photochemical injury. Horizontal lines in the dot-density plots indicate mean values. n = 10 mice/group. *P < 0.05; **P < 0.01, 1-way ANOVA followed by Tukey’s test. (E) Samples of human liver and omental white adipose tissue (WAT) from the subjects described in Supplemental Table 1 were assayed for SERPINE1 mRNA, and the plasma from these subjects was assayed for PAI-1 concentration. The graph shows plots of the indicated correlations, which were analyzed by linear regression, with the r2 and P values indicated in the graph. (F–I) Serpine1fl/fl mice were fed a high-fat diet for 4 months and then injected intravenously with AAV8-TBG-Cre (Cre) to target the PAI-1 gene Seprine1 in hepatocytes or with control AAV8-TBG-LacZ (LacZ). After 4 weeks, the following parameters were measured: (F) liver Serpine1 mRNA and plasma PAI-1 protein concentration; (G) plasma tPA activity measured by enzymatic assay; (H) plasma fibrinolytic activity measured by the euglobulin clot-lysis assay; and (I) tail-bleeding time and time to occlusive carotid arterial thrombosis induced by rose bengal/laser photochemical injury. n = 11 mice/group. *P < 0.05; **P < 0.01, 2-tailed Student’s t test.
Fig 5: Each group was assessed in triplicates except control groups and standards were assessed in duplicates (n = 3). The values of p <.05 were considered significant whereas * indicates p‐value <.05, **p‐value <.01 and ***p‐value <.001. This experiment was performed twice and independently. (A) The levels of tPA concentration in MCF‐7: Silencing of ANXA2 or EpCAM led to a significant increase in the concentration of tPA in the cell culture supernatants, compared to the control. The concentration of tPA in the cell culture supernatants increased and magnified upon silencing both ANXA2 and EpCAM in the double siRNA group. (B) The levels of tPA concentration in ZR‐75‐1: siRNA‐ANXA2 or siRNA‐EpCAM resulted in a significant increase in the concentration of tPA in the cell culture supernatants, compared to the control. The concentration of tPA in the cell culture supernatants increased and magnified due to silencing both ANXA2 and EpCAM in the double siRNA group. (C) The levels of tPA concentration in MDA‐MB‐231 and Hs578T: In both cell types, the concentration of tPA in cell culture supernatants increased significantly due to siRNA‐ANXA2 treatment
Supplier Page from Abcam for Tissue type Plasminogen Activator Activity Assay Kit (Colorimetric, Human)