Improved liver function and regeneration with A20

A20 改善肝功能和再生

• 批准号: 6570013
• 负责人: CHRISTIANE FERRAN
• 金额: $ 35.63万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6570013
• 关键词: Adenoviridae; apoptosis; cell line; cell proliferation; cysteine endopeptidases; enzyme activity; gene expression; hepatocyte growth factor; laboratory mouse; laboratory rat; lipopolysaccharides; liver cells; liver failure; liver function; liver metabolism; liver regeneration; mutant; necrosis; nuclear factor kappa beta; protein structure function; transcription factor; transfection /expression vector; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): Necrosis and apoptosis of hepatocytes are critical pathologic features associated with liver injury. Hepatocyte apoptosis is a feature of viral hepatitis, ischemic liver injury, sepsis, cholestasis, and a result of exposure to hepatotoxic substances such as ethanol, acetaminophen and cytostatic drugs. Massive hepatocyte apoptosis and necrosis result in fulminant hepatic failure (FHF). Only 14% of patients diagnosed with FHF recover with medical therapy. Orthotopic liver transplantation (OLT) has dramatically improved the fate of these patients (49% undergo OLT), yet 37% die while awaiting OLT. This gloomy picture is balanced by the unique capacity of the liver to regenerate. Hepatocyte replication leads to a full recovery of liver function and mass 1-2 weeks following surgical, viral or chemical hepatic loss. We propose that protecting hepatocytes from apoptosis and promoting their proliferation are two strategies that could beneficially impact FHF. Our preliminary data demonstrate that A20 promotes hepatocyte proliferation and is anti-apoptotic. A20 is part of the physiologic response of hepatocytes to injury. A20 is upregulated in hepatocytes by pro-inflammatory stimuli including TNF and LPS and functions to protect from TNF mediated apoptosis. Gene transfer of A20 to mice livers protects from lethality in the galactosamine and LPS (D-gal/LPS) model of toxic FHF. Adenovirus mediated expression of A20 in livers of BALB/c mice yields an 89% survival rate following administration of D-gal/LPS as compared to 15-20% in control mice. Mice expressing A20 maintain normal liver function as assessed by prothrombin time while controls suffer from a severe bleeding diathesis. Expression of A20 in the liver protects from lethality associated with a subtotal (87%) liver resection (LR). In this model, resection of 87% of the liver mass results in 100% lethality. In contrast, >60% of mice expressing A20 survive the 87% LR and demonstrate increased regenerative capacity as assessed by the number of PCNA (proliferating cell nuclear antigen) positive nuclei in the liver. These results qualify A20 as a critical gene involved in accelerating liver regeneration and promoting hepatocyte survival and function, even when facing extreme metabolic demands. These encouraging results prompted the submission of this proposal. Our specific aims are i) to dissect, in vitro, the molecular basis of the (1) anti-apoptotic and (2) pro-proliferative function of A20 in hepatocytes and ii) to confirm that liver directed gene therapy using A20 will beneficially impact upon toxic, FAS-mediated and surgical experimental models of FHF. From a basic science standpoint, the in vitro work proposed will address the effect of A20 upon transcription factors and expression of genes involved in apoptosis, activation and proliferation of hepatocytes. This should unveil many unknowns in our understanding of hepatocyte biology and could lead to the discovery of novel therapeutic targets. From a therapeutic standpoint, validation of the beneficial effect of A20 in the murine in vivo models of FHF should set the basis for extending this approach to models of FHF in non human primates and potentially to clinical applications. The generation of novel safer and tissue specific viral vectors for gene transfer and the development of non-viral means of protein delivery to cells will facilitate clinical translation of A20 based therapies.

描述（由申请人提供）：肝细胞的坏死和凋亡是与肝损伤相关的关键病理特征。肝细胞凋亡是病毒性肝炎，缺血性肝损伤，败血症，胆汁淤积的特征，也是暴露于肝毒性物质（例如乙醇，乙酰氨基氨基酚和细胞抑制药物）的结果。大量肝细胞凋亡和坏死导致肝衰竭（FHF）。只有14％的患有通过药物治疗诊断为FHF的患者。原位肝移植（OLT）显着改善了这些患者的命运（49％发生OLT），但在等待OLT时死亡37％。这张令人沮丧的画面与肝脏再生的独特能力平衡。肝细胞复制导致手术，病毒或化学肝损失后1-2周的肝功能完全恢复。我们建议保护肝细胞免受细胞凋亡的影响并促进其增殖是两种可能对FHF产生有益的策略。我们的初步数据表明，A20促进了肝细胞增殖，并且是抗凋亡。 A20是肝细胞对损伤的生理反应的一部分。 A20在肝细胞中通过包括TNF和LPS（LPS）以及功能的肝细胞上调，以防止TNF介导的凋亡。 A20向小鼠肝脏的基因转移可防止有毒FHF的半乳糖胺和LPS（D-GAL/LPS）模型中的致死性。腺病毒介导的A20在BALB/c小鼠肝脏中的表达在给药后，D-GAL/LP的给药后的存活率为89％，而对照小鼠的腺病毒症状为15-20％。表达A20的小鼠维持正常的肝功能，如凝血酶原时评估，而对照患有严重的出血素质。 A20在肝脏中的表达可免受与小计（87％）肝切除（LR）相关的致死性。在此模型中，切除87％的肝脏质量会导致100％的致死性。相比之下，表达A20的小鼠中有60％的LR生存，表现出增加的再生能力，如肝脏中PCNA（增殖细胞核抗原）阳性核评估所评估。这些结果符合A20的限定，即使面对极端的代谢需求，也可以促进肝脏再生并促进肝细胞的生存和功能。这些令人鼓舞的结果促使该提案提交。我们的具体目的是i）在体外剖析（1）抗凋亡的分子基础和（2）A20在肝细胞中的促生殖功能和ii）确认使用肝脏使用A20的肝脏基因治疗对有毒，FAS介导的和外科手术实验模型的有益影响。从基础科学的角度来看，提出的体外工作将解决A20对肝细胞凋亡，激活和增殖涉及的基因的转录因子的影响。这应该在我们对肝细胞生物学的理解中揭示许多未知数，并可能导致发现新颖的治疗靶标。从治疗的角度来看，在FHF的鼠体内模型中，A20对A20的有益效应的验证应为将这种方法扩展到非人类灵长类动物的FHF模型，并可能对临床应用。用于基因转移的新型更安全和组织特异性病毒载体的产生以及向细胞递送蛋白质递送的非病毒手段的发展将促进基于A20的疗法的临床翻译。