Molecular Determinants of liver sinusoidal endothelial cells for hepatic regeneration

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ABSTRACT End-stage liver disease is associated with morbidity and mortality often requiring a liver transplant. Dysfunction and capillarization of liver sinusoidal endothelial cells (LSECs) could contribute to impaired hepatic repair and cirrhosis. Thus, uncovering the mechanisms by which LSECs acquire their specialized functions to support hepatic repair could enable the development of therapies for scar-free liver repair. We have shown that activation of Id1 and Cxcr7 in LSECs induce angiocrine factors, that guide hepatic regeneration without fibrosis. However, the mechanism by which LSECs acquire their pro-hepatic functions is unknown. We show that while transcription factors (TFs) Fli1 and Erg dictate the vascular fate and homeostasis of LSECs (Gomez- Salinero JM, et al, Nature Cardiovascular Research, 2022), induction of TF c-Maf specifies the phenotype and regenerative functions of LSECs (Gomez-Salinero JM, et al, Cell Stem Cell, 2022). Induction of c-Maf in generic human endothelial cells (ECs) switches on liver-specific LSEC signatures and angiocrine factors supporting hepatocyte functionality. In mice in which c-Maf is deleted in adult ECs, recovery from CCl4 results in fibrosis and LSECs regression to arterial cell fate. Thus, we hypothesize that maintenance of LSEC vascular cell fate requires constitutive Fli1 or Erg expression, sustaining LSECs homeostatic functions. Induction of c- Maf enforces specialized pro-regenerative functions and interactions of LSECs with hepatocytes, Kupffer and stellate cells that prevents stress-induced LSEC arterialization promoting hepatic repair without fibrosis. To this end, we have reprogrammed human generic ECs to an adaptable tubulogenic state. These Reset-Vascular Endothelial Cells (R-VECs) self-assemble into a 3D vascular network, transporting human blood and arborizing hepatocytes (Palikuqi B et al. Nature, 2020). The vascularized hepatic aggregates with stellate and Kupffer cells within scalable and perfusable microfluidic devices establish a human Hepatic-on-VascularNet platform, enabling study of physiologically adaptive cross-talk between human hepatocytes and LSECs. This hypothesis will be tested by performing these Aims: Aim 1: Define the mechanism by which hierarchical Fli1 and Erg expression through c-Maf induction sustains specialization of LSECs at steady state, during hepatic regeneration and after CCl4 induced liver injury. AIM 2: Uncover the contribution of c-Maf expressed in the Kupffer cells that by enforcing and sustaining LSEC vascular attributes regulate hepatic homeostasis during liver regeneration and CCl4 induced liver injury. AIM 3: Employ the human Hepatic-On-VascularNet platform to uncover the mechanism by which c-Maf is induced functionally in human liver ECs to form specialized LSECs. Determine whether human c-Maf induced LSECs (iLSECs) can restore hepatic regeneration. Specifically, the role of infusing human iLSECs in restoring hepatic repair post-acetaminophen (APAP) injury without provoking fibrosis will be assessed. These studies will uncover the molecular determinants of human LSECs and allow strategies to employ iLSECs to sustain its pro-regenerative and anti-fibrotic attributes for liver repair.