Regulation of HNF4 in Hepatic Failure in Cirrhosis

描述（由申请人提供）：肝硬化肝功能衰竭的原因尚不清楚，因为肝脏有再生能力，尽管失去了一半以上的肝细胞，但功能正常。因此，肝硬化的肝功能衰竭是由剩余肝细胞的额外功能障碍引起的。我们的初步研究表明，来自具有失代偿功能的肝硬化肝脏的原代肝细胞在基因表达和增殖能力方面表现出许多改变。然而，尽管端粒缩短和端粒酶活性降低，这些肝细胞在移植到正常肝脏后，最终可以恢复其再生和功能的能力。我们的转录组分析表明，肝细胞衰竭的进展与转录因子HNF4¿的下调和其调节网络的抑制有关。由于HNF4¿缺乏可以解释下游效应物、成熟肝细胞特异性基因和一般肝功能的抑制，我们研究了培养的终末期肝硬化肝细胞，发现通过AAV转导恢复HNF4¿表达，显著并立即纠正了表型。然后，AAV-HNF4¿IV给失代偿性肝硬化啮齿动物。肝细胞功能在2周内恢复到正常水平，生存期从2周左右延长到100多天！因此，HNF4转录激活的破坏似乎是导致晚期肝硬化肝功能衰竭的机制。因此，我们假设恢复HNF4¿将通过纠正代谢缺陷和逆转肝细胞复制性衰老有效地治疗肝硬化终末期肝衰竭患者。在这些研究中，我们将使用重组AAV载体，编码由诱导启动子驱动的HNF4¿，以确定短期治疗HNF4¿是否可以导致肝功能和生存的持续正常化。此外，我们将评估HNF4¿-AAV治疗在CCL4持续损伤中的效用，以确定这种治疗的潜在临床疗效。为了确定HNF4下调在失代偿肝硬化中的机制，我们将对分离肝细胞和全肝进行全基因组ChIP-Seq分析。最后，我们将确定人类终末期肝硬化肝脏在多大程度上与我们在啮齿动物研究中发现的肝脏具有相同的特征，并将确定HNF4¿表达恢复可以使这些终末期肝脏衍生的人类肝细胞正常化的程度。

DESCRIPTION (provided by applicant): The cause of liver failure in cirrhosis is not well understood, as the liver is capable of regeneration and functions normally despite loss of more than half of its hepatocytes. Hepatic failure in cirrhosis therefore results from additional dysfunction of the remaining hepatocytes. Our Preliminary Studies showed that primary hepatocytes derived from cirrhotic livers with decompensated function exhibit numerous alterations in gene expression and proliferative capacity. Yet, despite critical shortening of telomeres and loss of telomerase activity, these hepatocytes can eventually recover their capacity for regeneration and function after transfer into a normal liver. Our transcriptome analysis demonstrated that progression to hepatocyte failure was associated with down regulation of transcription factor HNF4¿ and suppression of its regulatory network. Since HNF4¿ deficiency could explain the depression of downstream effectors, mature hepatocyte-specific genes, and general hepatic function, we studied end-stage cirrhotic hepatocytes in culture and found that HNF4¿ expression, restored via AAV transduction, dramatically and immediately corrected the phenotype. Next, AAV-HNF4¿ was given IV to rodents with decompensated cirrhosis. Their hepatocyte function improved to almost normal levels within 2 weeks and survival was prolonged from ~2 weeks to more than 100 days! Thus, disruption of HNF4¿ transcriptional activation appears to be the mechanism responsible for hepatic failure in advanced cirrhosis. We therefore hypothesize that restoration of HNF4¿ will effectively treat cirrhotic patients with end-stage liver failure by correcting the metabolic defects and reversing the hepatocyte replicative senescence. In these studies we will use a recombinant AAV vector that encodes HNF4¿ driven from an inducible promoter to determine whether short term treatment with HNF4¿ can lead to sustained normalization of hepatic function and survival. In addition, we will assess the utility of HNF4¿-AAV treatment during continuing injury with CCL4 to determine the potential clinical efficacy of such therapy. To determine the mechanism of HNF4¿ downregulation in decompensated cirrhosis, we will perform a genome-wide ChIP-Seq analysis of isolated hepatocytes and whole liver. Finally, we will determine the extent to which human end-stage cirrhotic livers share the same characteristics as those identified in our rodent studies, and will determine the extent that restoration of HNF4¿ expression can normalize human hepatocytes derived from these end-stage livers.