Fig 1: PF4 improves endothelial cell function by combining with heparin sulfate(A and B) After incubated with HSase (1 mU/ml, Sigma-Aldrich) for 4 h, cells were treated with 1 μM AngII or 5 μM TNFα alone, AngII or TNFα plus PF4 (10 μM) for 48 h, Cell adhesion was measured using CCK8 assay.(C and D) After incubated with HSase (1 mU/ml, Sigma-Aldrich) for 4 h, cells were treated with 1 μM AngII or 5 μM TNFα alone, AngII or TNFα plus PF4 (10 μM) for 36 h, treated cells were seeded onto the upper chamber for 12 h, cells on the lower side of the filter were detected using an inverted microscope.(E and F) After incubated with HSase (1 mU/ml, Sigma-Aldrich) for 4 h, cells were treated with 1 μM AngII or 5 μM TNFα alone, AngII or TNFα plus PF4(10 μM) for 36 h, treated cells were seeded onto the matrigel for 4 h in a 24-well plate. After incubating with 2 μM calcein AM for 30 min, the vascular cyclization ability of cells is detected. Data are expressed as mean ± SD. ∗p < 0.05, ∗ ∗p < 0.01, ∗ ∗ ∗p < 0.001.
Fig 2: Liposome-PF4 nanoparticles repress thoracic aortic dissection formation by improving endothelial function(A) Mice were treated with liposome-PF4, PF4, or empty liposome once every three days at 4 weeks. Three days after the first administration, mice were treated with 0.25% BAPN (β-aminopropionitrile monofumarate) for 21 days. Subsequently, mice were treated with 1 μg/kg per minute AngII using minipump (n = 10, per group).(B and C) Representative images of mice aorta were shown.(D and E) maximum aortic diameter was measured (n = 10, per group).(F) AD incidence was statistically analyzed.(G) Representative images of mouse aorta stained with CD31 (red) and DAPI (blue).(H) Effect of PF4 on MMP activities in cultured supernatant of vascular wall endothelial cells by gelatin zymography.(I) Representative macroscopic images of aorta sections were stained with hematoxylin and eosin (H&E), Masson, and elastic-Van Gieson (EVG) staining.(J) Quantification of elastin integrity in each group of mouse aortas. Data are expressed as mean ± SD. ∗p < 0.05, ∗ ∗p < 0.01, ∗ ∗ ∗p < 0.001.
Fig 3: Liposome encapsulation treatment reduces the degradation rate of PF4 protein in mice(A) PF4-SOD liposome was made through the rotation-evaporation method. Its morphology and diameter were detected using an electronic microscope.(B) Detection of liposome particle size using dynamic light scattering method.(C) ELISA assay for detecting the encapsulation efficiency of liposomes.(D) 100 μg/kg PF4 or PF4 liposome was injected into 8-week-old C57 mice through the tail vein, and the concentration of PF4 in the mouse blood was detected using ELISA assay at different time points.(E) After 100 μg/kg PF4 liposome-CY5 was injected into 8-week-old C57 mice for 12 h through the tail vein, the PF4 liposome-CY5 distribution was detected by immunofluorescence. Data are expressed as mean ± SD. ∗p < 0.05, ∗ ∗p < 0.01, ∗ ∗ ∗p < 0.001.
Fig 4: PF4 represses MAPK signal transduction by blocking FGFs-FGFR(A and B) Human aortic endothelial cells were treated with 1 μM AngII or 5 μM TNFα alone, 1 μM AngII or 5 μM TNFα plus PF4 at the indicated concentrations for 24 h. The phosphorylated and total levels of Erk1/2, P38, Akt (Ser 473), and Akt (Thr 308) were detected by western blotting analysis (n = 3).(C and D) Cells were treated with 1 μM AngII or 5 μM TNFα alone, AngII or TNFα plus PF4(10 μM) and SUN11602(10 μM) for 24 h. The phosphorylated and total levels of Erk1/2, P38, and Akt (Ser 473) were detected by western blotting analysis (n = 3). Density ratios of phosphorylated proteins to total proteins were presented as the mean ± standard deviation. ∗p < 0.05, ∗ ∗p < 0.01, ∗ ∗ ∗p < 0.001.
Fig 5: PF4 represses thoracic aortic dissection formation and improves endothelial cell function by inhibiting the activation of FGF-FGFR(A) Differential gene analysis between BAPN group and Lipo-PF4 group. Mice were treated with liposome-PF4 once every three days at 3 weeks. Three days after the first administration, mice were treated with 0.25% BAPN (β-aminopropionitrile monofumarate) for 14 days (n = 9, per group).Mouse aortic intima were isolated, and the aortic intima of three mice were mixed into one sample for transcriptome sequencing, and performed differential gene analysis. N: BAPN group; P: lipo-PF4+BAPN group.(B) GO analysis of differential genes.(C) Mice were treated with liposome-PF4 or liposome-PF4 plus SUN11602 once every three days at 3 weeks. Three days after the first administration, mice were treated with 0.25% BAPN (β-aminopropionitrile mono fumarate) for 25 days (n = 10, per group).(D) Representative images of mice aorta were shown.(E) Maximum aortic diameter was measured (n = 10, per group).(F) AD incidence was statistically analyzed.(G) Effect of PF4 and SUN11602 on MMP activities in cultured supernatant of vascular wall endothelial cells by gelatin zymography.(H) Representative macroscopic images of aorta sections were stained with hematoxylin and eosin (H&E), Masson, and elastic-Van Gieson (EVG) staining.(I) Quantification of elastin integrity in each group of mouse aortas.(J) Representative images of mouse aorta stained with CD31 (red) and DAPI (blue). Data are expressed as mean ± SD. ∗p < 0.05, ∗ ∗p < 0.01, ∗ ∗ ∗p < 0.001.
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