Melatonin attenuates splanchnic hyperdynamic circulation in cirrhotic portal hypertension by disrupting POMK-VEGFR2 interaction

Figure 1 Melatonin alleviated bile duct ligation-induced liver cirrhosis and portal hypertension. A: Construction of animal model of liver cirrhosis and melatonin administration; B: The levels of plasma liver injury markers alanine aminotransferase, aspartate aminotransferase and total bilirubin in rats; C: The histological staining of liver tissue sections using hematoxylin and eosin, Masson’s trichrome, and Sirius red. Immunohistochemical staining of collagen I and α-smooth muscle actin in rat livers; D: Quantitative analysis of histopathological and immunohistochemical staining in C; E: Western blotting of hepatic VEGFR-2 and its phosphorylated form; F: Quantitative analysis of VEGFR-2 phosphorylation. ^aP < 0.05. ^bP < 0.01. BDL: Bile duct ligation; MLT: Melatonin; α-SMA: Α-smooth muscle actin; HE: Hematoxylin and eosin; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase.

Figure 2 Melatonin alleviated bile duct ligation-induced mesenteric vascular remodeling and angiogenesis. A: Representative hematoxylin and eosin staining images of mesenteric artery. The yellow arrows represent the thickness of vascular wall; B: Quantitative analysis of the wall thickness; C: Enzyme-linked immunosorbent assay of inflammatory cytokines in mesenteric artery tissues; D: Representative immunohistochemical images of CD68 in the mesentery; E: Quantitative analysis of CD68-positive cells; F: Representative immunohistochemical images of CD31 in the mesentery; G: Quantitative analysis of CD31 staining intensity; H: Western blotting of mesenteric VEGFR-2 and its phosphorylated form; I: Quantitative analysis of VEGFR-2 phosphorylation. ^aP < 0.05. ^bP < 0.01. BDL: Bile duct ligation; MLT: Melatonin; HE: Hematoxylin and eosin; IL: Interleukin; TNF-α: Tumor necrosis factor alpha.

Figure 3 RNA-sequencing of mesenteric arteries from bile duct ligation rats treated with melatonin. A: The volcano plot of differential expressed genes (DEGs) between bile duct ligation (BDL) and BDL + melatonin (MLT) groups; B: The heatmap of DEGs between BDL and BDL + MLT groups; C: The protein-protein interaction network of DEGs with 10 hub genes; D: The top 10 terms enriched by Gene Ontology enrichment analysis based on DEGs; E: The top 10 pathways enriched by Kyoto Encyclopedia of Genes and Genomes analysis based on DEGs; F: The expression profile of VEGFs between BDL and BDL + MLT groups; G: The validation of the expression levels of VEGFs by quantitative real-time polymerase chain reaction. ^aP < 0.05. ^bP < 0.01. BDL: Bile duct ligation; MLT: Melatonin.

Figure 4 VEGFR-2 inhibition alleviated bile duct ligation-induced cirrhotic portal hypertension. A: Construction of animal model of liver cirrhosis and AZD2171 administration; B: The portal hypertension hemodynamic parameters in rats from 4 groups; C: The histological staining of liver tissue sections using hematoxylin and eosin, Masson’s trichrome, and Sirius red. Immunohistochemical (IHC) staining of Collagen I and α-smooth muscle actin; in rat livers; D: Quantitative analysis of histopathological and IHC staining in C; E: Representative IHC images of CD68 in the mesentery; F: Quantitative analysis of CD68-positive cells; G: Representative IHC images of CD31 in the mesentery; H: Quantitative analysis of CD31 staining intensity. ^aP < 0.05. ^bP < 0.01. α-SMA: Α-smooth muscle actin; HE: Hematoxylin and eosin; BDL: Bile duct ligation.

Figure 5 Melatonin and AZD2171 inhibited angiogenesis in vitro. A: Western blotting of VEGFR-2 and its phosphorylated form in human umbilical vein endothelial cells (HUVECs) treated with VEGFA, melatonin (MLT), or AZD2171; B: Quantitative analysis of VEGFR-2 phosphorylation; C: The proliferation assay of HUVECs treated with VEGFA, MLT, or AZD2171; D: The tube formation assay of HUVECs treated with VEGFA, MLT, or AZD2171; E: Quantitative analysis of tube formation assay; F: The wound healing assay of HUVECs treated with VEGFA, MLT, or AZD2171; G: Quantitative analysis of wound healing assay; H: Transwell migration assay of HUVECs treated with VEGFA, MLT, or AZD2171; I: Quantitative analysis of Transwell migration assay. ^aP < 0.05. ^bP < 0.01. MLT: Melatonin.

Figure 6 Melatonin suppressed VEGF signaling and interactions between VEGFR-2 and POMK. A: Western blotting of VEGFR-2 signaling in human umbilical vein endothelial cells (HUVECs) treated with VEGFA, melatonin, or AZD2171; B: Quantitative analysis of the activation of VEGFR-2 signaling; C: Immunoprecipitation (IP) blotting of VEGFR-2 in HUVECs and subjected of IP-mass spectrometry; D: PPI network of enriched proteins by IP-mass spectrometry; E: IP blotting of DOCK5 by VEGFR-2 in HUVECs; F: IP blotting of DSG1 by VEGFR-2 in HUVECs; G: IP blotting of POMK by VEGFR-2 in HUVECs; H: Quantitative analysis of IP blotting. ^aP < 0.05. ^bP < 0.01. MLT: Melatonin.

Figure 7 Melatonin suppressed VEGFR-2 signaling via a POMK-dependent manner. A: Validation of VEGFR-2 knockdown by siRNA in human umbilical vein endothelial cells (HUVECs); B: Western blotting of VEGFR-2 signaling in HUVECs with VEGFR-2 knockdown; C and D: Quantitative analysis of PLCγ and ERK phosphorylation in B; E: Validation of POMK knockdown by siRNA in HUVECs; F: Western blotting of VEGFR-2 signaling in HUVECs with POMK knockdown; G-I: Quantitative analysis of VEGFR-2, PLCγ, and ERK phosphorylation in F. ^bP < 0.01. MLT: Melatonin.

Figure 8 Proposed mechanism underlying the protective effects of melatonin against pathological angiogenesis in cirrhotic portal hypertension. Melatonin disrupts the interaction between POMK and VEGFR-2, resulting in reduced VEGFR-2 phosphorylation and attenuation of downstream PLC-γ/ERK1/2 signaling activation. This inhibition suppresses VEGF-induced endothelial cell proliferation and angiogenesis, thereby contributing to the improvement of splanchnic hyperdynamic circulation in cirrhotic portal hypertension. HUVEC: Human umbilical vein endothelial cell.