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有关，通常需要肝移植。功能障碍 而肝窦内皮细胞(LSECs)的毛细血管化可能有助于受损的肝脏修复和 肝硬变。因此，揭示了LSEC获得其专门功能以支持 肝修复可以使无疤痕肝修复疗法的发展成为可能。我们已经证明了 在LSECs中Id1和CXCR7的激活诱导血管分泌因子，这些因子引导肝再生而不是 纤维化症。然而，LSECs获得其促肝功能的机制尚不清楚。我们展示了 转录因子(TF)FlI1和Erg决定LSECs的血管命运和动态平衡。 Salineo JM等人，自然心血管研究，2022)，Tf c-Maf的诱导指定了表型和 LSECs的再生功能(Gomez-Salineo JM等，Cell Stem Cell，2022)。原癌基因c-Maf的诱导作用 通用人内皮细胞(ECs)开关肝脏特异性LSEC信号和血管分泌因子 支持肝细胞功能。在成年内皮细胞c-Maf缺失的小鼠中，从CCl4中恢复的结果 纤维化和LSECs回归到动脉细胞的命运。因此，我们假设LSEC血管的维持 细胞命运需要组成性的FLI1或ERG表达，维持LSECs的动态平衡功能。C-的诱导 MAF增强LSECs的特殊促再生功能以及与肝细胞、Kupffer和 星状细胞，防止应激诱导的LSEC动脉化，促进肝修复，无纤维化。对这件事 最后，我们对人类通用内皮细胞进行了重新编程，使其处于一种适应性强的小管生成状态。这些重置血管 血管内皮细胞(R-VECs)自组装成3D血管网络，运输人体血液和树枝 肝细胞(Palikuqi B et al.自然，2020)。带血管的肝星状和枯否集合体 可扩展和可灌流的微流控设备内的细胞建立了人类肝脏血管网平台， 能够研究人肝细胞和LSECs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：定义层级FLI1 C-Maf诱导的ERG表达支持LSECs在稳定状态下的特化 再生和CCl4诱导的肝损伤后。目标2：揭示c-Maf在 Kupffer细胞通过强制和维持LSEC血管属性来调节肝脏的动态平衡 肝再生和CCl4诱导的肝损伤。目的3：利用人类肝血管网平台 揭示c-Maf在人肝内皮细胞中功能诱导形成特化LSECs的机制。 确定人c-Maf诱导的LSECs(ILSECs)是否能恢复肝再生。具体地说， 人iLSECs输注对扑热息痛(APAP)后肝损伤的修复作用 将对纤维化进行评估。这些研究将揭示人类LSEC的分子决定因素，并允许 利用iLSECs维持其促再生和抗肝纤维化的肝修复特性的策略。

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.