Molecular Determinants of liver sinusoidal endothelial cells for hepatic regeneration

摘要 终末期肝病与发病率和死亡率相关，通常需要肝移植。功能障碍 肝窦内皮细胞（LSECs）的毛细血管化可能导致肝脏修复受损， 肝硬化因此，揭示LSEC获得其专门功能以支持 肝修复可以使无瘢痕肝修复疗法的发展成为可能。我们已经证明 LSEC中Id 1和Cxcr 7的激活诱导血管分泌因子，其引导肝再生，而不 纤维化然而，LSEC获得其促肝功能的机制尚不清楚。我们表明 虽然转录因子（TF）Fli 1和Erg决定了LSEC的血管命运和体内平衡（Gomez， Salinero JM等人，Nature Cardiovascular Research，2022），TF c-Maf的诱导指定了表型， LSEC的再生功能（Gomez-Salinero JM等人，Cell Stem Cell，2022）。诱导c-Maf在 普通人内皮细胞（EC）对肝脏特异性LSEC特征和血管分泌因子的开关 支持肝细胞功能。在成年EC中c-Maf缺失的小鼠中，从CCl 4中恢复导致 在纤维化和LSECs回归到动脉细胞命运中。因此，我们假设维持LSEC血管 细胞命运需要组成型Fli 1或Erg表达，维持LSEC稳态功能。诱导C- Maf执行专门的促再生功能以及LSEC与肝细胞、Kupffer和 防止应激诱导的LSEC动脉化的星状细胞，促进肝修复而无纤维化。本 最后，我们已经重新编程人类通用EC适应tubulogenic状态。这些复位-血管 内皮细胞（R-VEC）自组装成3D血管网络，运输人体血液并形成树枝状结构。 肝细胞（Palikuqi B等人，Nature，2020）。血管化的肝聚集体伴星状和枯否细胞 在可扩展和可灌注的微流体装置中的细胞建立了人肝血管网平台， 使得能够研究人肝细胞和LSEC之间的生理适应性串扰。这 将通过执行以下目标来检验假设：目标1：定义分层Fli 1 通过c-Maf诱导的Erg表达维持了稳态下LSEC的特化， 再生和CCl_4诱导的肝损伤。目的2：揭示c-Maf表达在肿瘤细胞中的作用。 库普弗细胞通过加强和维持LSEC血管属性来调节肝脏稳态， 肝再生和CCl 4诱导的肝损伤。目的3：采用人类肝脏血管网平台， 揭示c-Maf在人肝EC中功能性诱导形成特化LSEC的机制。 确定人c-Maf诱导的LSEC（iLSEC）是否可以恢复肝再生。具体而言是 输注人iLSEC在恢复对乙酰氨基酚（APAP）损伤后肝修复中的作用， 将评估纤维化。这些研究将揭示人类LSEC的分子决定因素， 使用iLSEC来维持其用于肝修复的促再生和抗纤维化属性的策略。

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.