Investigation of the role of VEGFA in harnessing cholangiocyte-driven liver regeneration

项目总结/摘要 肝病影响着全球数亿人。肝移植仍然是唯一的治疗方法 终末期肝病（ESLD）;然而，由于肝脏短缺，移植面临着重大负担 捐助者。众所周知，肝脏通过肝细胞驱动的再生具有显著的再生能力， 主要功能性肝细胞类型肝细胞的增殖。然而，在ESLD中，肝细胞增殖 变得筋疲力尽治疗ESLD的另一种策略是通过胆道刺激促进肝再生。 上皮细胞（BEC）驱动的再生作为一种新的机制，可以利用作为一个治疗目标。 BEC驱动的肝再生是BEC增殖、转化为肝祖细胞（LPC）的过程 阶段，定义为导管反应（DR）的过程，并分化为功能性肝细胞和恢复肝脏 功能BEC驱动的再生已经在小鼠模型中得到实验证明，其中肝细胞 在我们的斑马鱼模型中，在肝细胞几乎完全消融后，增殖受到损害。 在几乎所有慢性和急性人类肝病中观察到DR以及BEC出芽肝细胞 提示BEC驱动的修复发生在人类，但分化成肝细胞是不可能的。 不足以缓解肝脏疾病。因此，我们的目标是确定和测试一种药物途径， 触发BEC向肝细胞的转化，以有效地治疗性使用BEC来治疗肝病。我们 提出VEGFA促进BEC向肝细胞的转化，并在互补中拯救肝功能， 小鼠和斑马鱼肝损伤模型。我们的初步数据表明，VEGFA在受伤的人中的输送 通过与脂质纳米粒复合的非整合且安全的核苷修饰的mRNA对小鼠肝脏的影响 （mRNA-LNP）诱导BEC向肝细胞的稳健转化以及脂肪变性和纤维化的逆转。此外，委员会认为， 阻断VEGFR 2（VEGFA的主要受体）或下游介质PI 3 K/AKT可消除BEC驱动的 斑马鱼的肝再生，表明VEGFR 2/PI 3 K/AKT轴的关键贡献。以前的研究 显示VEGFA通过刺激内皮细胞（EC）上的VEGFR 2促进啮齿动物的肝脏修复， 诱导肝细胞有丝分裂原的分泌，从而驱动肝细胞驱动的修复。然而， 报道，VEGFA直接影响BEC驱动的修复，这是我们的初步数据所建议的 显示了在受损小鼠肝脏中BEC亚群中的VEGFR 2诱导，其他人也报道了这一观察结果。 受伤的老鼠因此，我们的研究结果结合其他人的研究，使我们能够使用 互补的小鼠和斑马鱼肝损伤模型：（目的1）体内VEGFA递送触发BEC-至- 肝细胞转化，以补充失去的细胞质量和恢复肝功能，（目的2）VEGFA的作用 直接作用于BEC和/或间接作用于表达VEGFR 2的EC。这项研究可能具有关键的临床意义 通过建立一种治疗方法来预防肝病的进展，通过利用替代的内在 通过使用临床安全的mRNA-LNP的BEC驱动的肝再生来增强肝脏的再生能力。

PROJECT SUMMARY / ABSTRACT Liver disease affects hundreds of millions of people worldwide. Liver transplantation remains the only treatment for end stage liver disease (ESLD); however, transplantation faces a major burden due to a shortage of liver donors. The liver is known for a remarkable regenerative ability through hepatocyte-driven regeneration, the proliferation of the main functional liver cell type, the hepatocyte. However, in ESLD, hepatocyte proliferation becomes exhausted. An alternative strategy to treat ESLD would be to promote liver regeneration through biliary epithelial cell (BEC)-driven regeneration as a novel mechanism that could be exploited as a therapeutic target. BEC-driven liver regeneration is a process by which BECs proliferate, transition to a liver progenitor cell (LPC) stage, a process defined as ductular reaction (DR), and differentiate into functional hepatocytes and restore liver function. BEC-driven regeneration has been experimentally demonstrated in mouse models in which hepatocyte proliferation was compromised and in our zebrafish models after near complete ablation of hepatocytes. Observation of DR in virtually all chronic and acute human liver diseases and of budding hepatocytes from BECs in human cirrhotic livers suggests that BEC-driven repair occurs in human, yet differentiation into hepatocytes is insufficient to alleviate the liver disease. Our goal is therefore to identify and test a druggable pathway that triggers BEC-to-hepatocyte conversion for the efficient therapeutic use of BECs to treat liver diseases. We propose that VEGFA promotes BEC-to-hepatocyte conversion and rescues liver function in complementary mouse and zebrafish liver injury models. Our preliminary data demonstrate that delivery of VEGFA in injured mouse livers via the non-integrative and safe nucleoside-modified mRNA complexed with lipid nanoparticles (mRNA-LNP) induces robust BEC-to-hepatocyte conversion and reversion of steatosis and fibrosis. Moreover, blocking VEGFR2, the main receptor for VEGFA, or downstream mediators PI3K/AKT abrogates BEC-driven liver regeneration in zebrafish, suggesting the key contribution of the VEGFR2/PI3K/AKT axis. Previous studies showed that VEGFA promotes liver repair in rodents by stimulating VEGFR2 on endothelial cells (ECs) that induce the secretion of hepatocyte mitogens and thus drives hepatocyte-driven repair. However, it has not been reported that VEGFA directly affects BECs for BEC-driven repair, which is suggested by our preliminary data showing VEGFR2 induction in a subset of BECs in injured mouse livers, an observation also reported by others in injured rats. Therefore, our findings combined with studies from others lead us to test 2 hypotheses using the complementary mouse and zebrafish liver injury models: (Aim 1) VEGFA delivery in vivo triggers BEC-to- hepatocyte conversion to replenish the lost cell mass and restore liver functions, and that (Aim 2) VEGFA acts directly on BECs and/or indirectly on ECs that express VEGFR2. This study may have key clinical significance by establishing a treatment to prevent progression of the liver disease, by exploiting the alternative intrinsic regenerative ability of the liver via BEC-driven liver regeneration using clinically safe mRNA-LNPs.