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

Hamda Siddiqui; Preety Rawal; Chaggan Bihari; Naveen Arora; Savneet Kaur

doi:10.1016/j.jceh.2019.11.011

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

J Clin Exp Hepatol. 2020 Jul-Aug;10(4):275-283. doi: 10.1016/j.jceh.2019.11.011. Epub 2019 Dec 10.

Authors

Hamda Siddiqui^{1

2}, Preety Rawal², Chaggan Bihari¹, Naveen Arora¹, Savneet Kaur¹

Affiliations

¹ Institute of Liver and Biliary Sciences, New Delhi, India.
² Gautam Buddha University, Greater Noida, Uttar Pradesh, India.

Abstract

Aim: 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).

Methods: 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 coculture studies. 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.

Results: Of all the angiogenic factors, the gene expression of VEGF and 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 in vitro 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.

Conclusion: Our study indicates an important contribution of VEGF toward the activation of HPC-mediated regeneration and DR in NASH.

Keywords: Ang2, angiopoietin 2; BSA, bovine serum albumin; CM, conditioned medium; DMEM, Dulbecco's Modified Eagle medium; DR, ductular reaction; ELISA, enzyme-linked immunosorbent assay; EpCAM, epithelial cell adhesion molecule; FBS, fetal bovine serum; H&E, hematoxylin and eosin; HPC, hepatic progenitor cell; HSC, hepatic stellate cell; HUVEC, human umbilical vein endothelial cell; IHC, immunohistochemical; MT, Masson trichrome; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PCR, polymerase chain reaction; VEGF, vascular endothelial growth factor; angiogenesis; ductular reaction; hepatic progenitor cells; nonalcoholic steatohepatitis.