Isolation and immortalization of human portal vein endothelial cells: a novel research tool for investigating splanchnic vasculature

Cirrhosis profoundly impacts extrahepatic vasculature, particularly altering the portal venous system (PVS), leading to increased portal pressure, portosystemic collaterals, and portal vein thrombosis, which heightens morbidity and reduced survival in patients with liver disease. Despite extensive research on intrahepatic vasculature in cirrhosis, understanding of extrahepatic changes in the splanchnic territory at the molecular level remains limited. The distinct characteristics of the PVS suggest a unique endothelial profile crucial for elucidating vascular dysfunction in liver diseases. However, current research is hampered by the inaccessibility of the portal vein and imperfect preclinical models. Here, we aim to isolate and immortalize primary human portal vein endothelial cells (hPVECs) to enhance understanding of pathophysiological changes during liver disease and establish a platform for future drug testing. Overall design: hPVECs and inferior cava vein (ICV) endothelial cells (ECs) were isolated from human portal vein or ICV, obtained during hepatic transplantation, using trypsinization, mechanical scratching, and FACS. Subsequently, hPVECs were immortalized (iPVECs) with lentiviral particles expressing the SV40 large T-antigen. iPVECs maintained endothelial phenotype and behavior along with specific endothelial gene expression profiles distinct from systemic ECs. We then expanded the obtained cells and performed bulk RNAseq with hPVEC and hICVEC passage 1 cells iPVEC passage 20 cells. Comparative gene expression analysis was performed between hICVEC vs hPVECs and also compared to iPVEC.