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

项目摘要/摘要 肝病影响着全球数亿人。肝移植仍是唯一的治疗方法 对于终末期肝病(ESLD)；然而，由于肝脏短缺，移植面临主要负担 捐赠者。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， 增殖的主要功能肝细胞类型为肝细胞。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促进肝脏再生。 上皮细胞(BEC)驱动的再生是一种新的机制，可以作为治疗的靶点。 BEC诱导的肝再生是BEC增殖转化为肝祖细胞(LPC)的过程 阶段，被定义为导管反应(DR)的过程，并分化为功能肝细胞并恢复肝脏 功能。BEC驱动的再生已经在小鼠模型中实验证明，在这些模型中，肝细胞 在我们的斑马鱼模型中，在几乎完全切除肝细胞后，增殖受到了损害。 几乎所有慢性和急性人类肝病和BECs出芽肝细胞中DR的观察 在人类肝硬变中的研究表明，在人类中发生了由bec驱动的修复，但分化为肝细胞是 不足以缓解肝病。因此，我们的目标是识别和测试一种可用药途径 触发BEC向肝细胞的转化，从而有效地利用BECs治疗肝病。我们 建议VEGFA促进BEC向肝细胞转化，在补体中挽救肝功能 小鼠和斑马鱼肝损伤模型。我们的初步数据显示，在受伤的人中，VEGFA的交付 非整合型、安全的核苷修饰信使核糖核酸与脂质纳米粒复合的小鼠肝脏 (mRNA-LNP)可诱导BEC向肝细胞的转化，逆转脂肪变性和纤维化。此外， 阻断VEGFR2，VEGFA的主要受体或下游介质PI3K/AKT可消除BEC驱动 斑马鱼的肝脏再生，提示VEGFR2/PI3K/AKT轴的关键作用。以前的研究 研究表明，VEGFA通过刺激内皮细胞(ECs)上的VEGFR2来促进啮齿动物的肝脏修复 诱导肝细胞有丝分裂原分泌，从而驱动肝细胞驱动的修复。然而，它并没有 报道称，VEGFA直接影响BEC进行BEC驱动的修复，这是我们的初步数据表明的 显示VEGFR2在受损小鼠肝脏的BECs亚群中诱导，其他人也报道了这一观察结果 在受伤的老鼠身上。因此，我们的发现与其他人的研究相结合，导致我们使用 互补性小鼠和斑马鱼肝损伤模型：(目的1)VEGFA体内注射触发BEC-to- 肝细胞转化以补充丢失的细胞团和恢复肝功能，以及(目标2)VEGFA的作用 直接在BEC和/或间接在表达VEGFR2的EC上表达。这项研究可能具有重要的临床意义。 通过建立一种治疗方法来防止肝病的进展，通过利用替代的内在 使用临床安全的mRNALNPs通过BEC驱动的肝再生来实现肝再生的能力。

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.