Platelet activation relieves liver portal hypertension via the lymphatic system though the classical vascular endothelial growth factor receptor 3 signaling pathway

Figure 1 Trends in the number of rat lymphatic vessels at 2 weeks and 4 weeks after bile duct ligation. A: Representative immunohistochemistry images of Masson’s trichrome, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), vascular endothelial growth factor C (VEGF-C) and VEGF receptor 3 (VEGFR-3) staining in the livers of the different groups; B: Quantitative analysis of Masson’s trichrome; C: Quantitative analysis of LYVE-1; D: Quantitative analysis of VEGF-C; E: Quantitative analysis of VEGFR-3 staining; F: Luminal area of lymphatic vessels were also measured; G: The number of lymphatic vessels was also measured. The data represent the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. BDL: Bile duct ligation; W: Week.

Figure 2 Intervention with adeno-associated virus-vascular endothelial growth factor C relieves liver fibrosis and portal hypertension by increasing the number of lymphatic vessels involved in bile duct ligation. A: Representative immunohistochemistry images of Masson’s trichrome, virus-vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor receptor 3 (VEGFR-3) and transforming growth factor beta (TGF-β) staining in the livers of the different groups; Quantitative analysis of B: Masson’s trichrome; C: VEGF-C; D: VEGFR-3; E: TGF-β staining; F: The levels of portal pressure were also measured. The data represent the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. AAV: Adeno-associated virus; BDL: Bile duct ligation; PP: Portal pressure; TGF-β: Transforming growth factor beta; VEGF-C: Vascular endothelial growth factor C; VEGFR-3: Vascular endothelial growth factor receptor 3; VEH: Vehicle.

Figure 3 Identification of the differentially expressed genes in the bile duct ligation lymphatic system and related biological processes. A: Heatmap comparison of differentially expressed genes (DEGs) in the lymphatic system in the bile duct ligation (BDL) and control groups; B: Volcano map of DEG expression in the lymphatic system between the BDL and control groups; C: Bubble plot for gene ontology enrichment of DEGs; D: Bubble plot for Kyoto encyclopaedia of genes and genomes enrichment of DEGs. BDL: Bile duct ligation; Con: Control.

Figure 4 Twelve core differentially expressed genes in the bile duct ligation lymphatic system and their related biological processes. A: Heatmap of 12 core differentially expressed genes (DEGs) in the lymphatic system between the bile duct ligation (BDL) and control groups; B: Volcano map of core DEGs in the lymphatic system between the BDL and control groups; C: Protein-protein interaction network between the DEGs and the 12 core DEGs; D: Co-expression of these core DEGs; E: Bar plot of the gene ontology enrichment of the core DEGs. BDL: Bile duct ligation; BP: Biological process; CC: Cellular component; Con: Control; MF: Molecular function.

Figure 5 Platelet-rich plasma-induced platelets increase intrahepatic lymphatic vessels via the vascular endothelial growth factor C/vascular endothelial growth factor receptor 3 pathway. A: Representative immunohistochemistry images of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor receptor 3 (VEGFR-3), fibronectin (FN) and cluster of differentiation 41 (CD41) in the livers of the different groups; Quantitative analysis of B: LYVE-1; C: VEGF-C; D: VEGFR-3; E: The luminal area; F: The number of lymphatic vessels were also measured; G: FN; H: CD41 expression. The data represent the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. BDL: Bile duct ligation; PRP: Platelet-rich plasma; VEH: Vehicle.

Figure 6 Inhibition of vascular endothelial growth factor C/vascular endothelial growth factor receptor 3 reversed the amelioration of liver fibrosis and portal hypertension in platelet-rich plasma-induced platelets. A: Representative immunohistochemistry images of hematoxylin and eosin (H&E), Masson’s trichrome, and Sirius red staining in liver samples from the different groups; Quantitative analysis of B: Masson’s trichrome; C: Sirius Red; D: Protein expression of alpha smooth muscle actin (α-SMA) in the different groups; Quantitative analysis of E: α-SMA staining; F: The levels of portal pressure were also measured. The data represent the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. BDL: Bile duct ligation; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; PP: Portal pressure; PRP: Platelet-rich plasma; VEH: Vehicle.

Figure 7 Inhibition of vascular endothelial growth factor C/vascular endothelial growth factor receptor 3 reverses the amelioration of intrahepatic angiogenesis and inflammation by platelet-rich plasma-induced platelets. A: Representative immunohistochemistry images of von Willebrand factor (vWF), matrix metalloproteinase 2 (MMP2), vascular endothelial growth factor A (VEGF-A), cluster of differentiation 31 (CD31) and CD68 in the livers of different groups; Quantitative analysis of B: vWF; C: MMP2; D: VEGF-A; E: CD31; F: CD68. The data represent the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. BDL: Bile duct ligation; PRP: Platelet-rich plasma; VEH: Vehicle.

Figure 8 Inhibition of vascular endothelial growth factor C/vascular endothelial growth factor receptor 3 reverses the amelioration of mesenteric angiogenesis and inflammation by platelet-rich plasma-induced platelets. A: Representative immunohistochemistry images of hematoxylin and eosin, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor receptor 3 (VEGFR-3) and cluster of differentiation 68 (CD68) in the mesentery of the different groups; Quantitative analysis of B: Wall thickness; C: LYVE-1; D: VEGF-C; E: VEGFR-3; F: CD68. The data represent the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. BDL: Bile duct ligation; PRP: Platelet-rich plasma; SO-VEH: Sham-operated-vehicle; VEH: Vehicle.