Panoply™ Human DLL4 Over-expressing Stable Cell Line

During the progression of liver fibrosis, liver sinusoidal endothelial cells (LSECs) undergo capillarization (i.e., loss of fenestrae) and produce basement membranes. DLL4, a ligand of the Notch signaling pathway, is primarily expressed in endothelial cells and maintains sinusoidal homeostasis. Here, researchers aimed to investigate the role of DLL4 in LSEC capillarization. They found that DLL4 is upregulated in human and CCl4-induced mouse fibrotic LSECs, consistent with LSEC capillarization and liver fibrosis. Primary mouse LSECs also underwent capillarization in vitro, accompanied by DLL4 overexpression. Bioinformatics analysis confirmed that DLL4 induces LSEC production of basement membrane proteins, and that basement membrane protein production was increased in LSECs from mice treated with CCl4 for 4 and 6 weeks. DLL4 overexpression also increased hepatic stellate cell (HSC) coverage of the sinusoids through endothelin-1 (ET-1) synthesis. Hypoxia is a key factor driving DLL4 overexpression in liver tubular epithelial cells (LSECs). Consistent with these findings, DLL4 silencing in vivo attenuates LSEC capillarization and CCl4-induced liver fibrosis. In summary, DLL4 mediates LSEC capillarization and the vicious cycle between fibrosis and pathological sinusoidal remodeling.

To investigate the role of DLL4 in hepatic vascular resistance, the researchers analyzed the expression levels of several genes associated with endothelial resistance in TMNK-1 cells (Figure 1A). Compared with the control group, DLL4 overexpression resulted in downregulation of VEGFR2, Tie1, and Tie2, and upregulation of VEGFR1 and ET-1, whereas VEGFR3, caveolin-1, and eNOS remained unchanged (Figure 1B). DLL4 knockdown had the opposite effect on these factors (Figure 1B). Western blotting further confirmed that DLL4 induced ET-1 expression (Figure 1C). Primary LSECs isolated from mice treated with CCl₄ for 4 and 6 weeks had low eNOS and p-eNOS levels, despite significant upregulation of ET-1 (Figure 1D). To determine the effects of DLL4-overexpressing LSECs on hematopoietic stem cells (HSCs), the researchers established a coculture system of TMNK-1 cells with LX-2 cells and assessed the migration rate of the latter. As shown in Figure 1E, LX-2 cells showed significantly increased migration toward DLL4-overexpressing TMNK-1 cells compared with control cells, whereas DLL4 knockdown inhibited HSC migration. Furthermore, after co-culture on Matrigel, LX2 cells formed robust, interconnected cord-like structures with DLL4-overexpressing TMNK-1 cells, and physical contact between LX2 cells and DLL4-knockdown TMNK-1 cells was reduced (Figure 1F).

Figure 1. DLL4 enhanced sinusoidal coverage by HSCs. (Chen L, et al., 2019)