Vascular Endothelial Growth Factor Promotes Proliferation of Epithelial Cell Adhesion Molecule–Positive Cells in Nonalcoholic Steatohepatitis

Abstract

An impaired hepatocyte proliferation during severe liver injury causes the proliferation of hepatic progenitor cells (HPCs), also called as the ductular reaction (DR). In the present study, we studied the role of key angiogenic factors in HPC-mediated DR in nonalcoholic steatohepatitis (NASH). Liver biopsies from patients with NASH (n= 14) were included in the study. Patients with NASH were divided in two groups, early and late fibrosis (based on fibrosis staging). Biopsies were used to analyze the gene expression by quantitative real-time polymerase chain reaction and immunohistochemical (IHC) staining for two markers of DR, viz, CK19 and epithelial cell adhesion molecule (EpCAM). Cocultures were performed between steatotic human umbilical vein endothelial cells (HUVECs) and LX2 and Huh7 cells. Enzyme-linked immunosorbent assays were performed to measure levels of vascular endothelial growth factor (VEGF) in coculturestudies. Next, Huh7 cells were treated with VEGF, and proliferation was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays. The number of EpCAM-positive cells was analyzed by flow cytometry. Of all the angiogenic factors, the gene expression of VEGFand angiopoietin 2 (Ang2) was significantly different between patients with NASH in the early and late fibrosis groups (P<0.05 for both). Both VEGF and Ang2 also correlated significantly with the IHC scores of CK19 and EpCAM in the study group. In the invitro studies, VEGF levels were significantly increased when Huh7 cells were cocultured with steatotic HUVECs and LX2 cells. The proliferation and percentage of EpCAM-positive cells was increased when Huh7 cells were treated with VEGF. Our study indicates an important contribution of VEGF towardthe activation of HPC-mediated regeneration and DR in NASH.