Caspase Dependent Cell Death in Nonalcoholic Fatty Liver Disease

描述（由申请人提供）：非酒精性脂肪性肝病（NAFLD）是全球慢性肝病的主要原因。据估计，它现在影响了近30%的美国成年人口。NAFLD代表广泛的病症，范围从一般遵循良性非进展性临床过程的单纯性脂肪肝到脂肪性肝炎或NASH，一种更严重形式的NAFLD，其特征在于肝细胞凋亡、肝损伤和炎症，其可能进展为肝硬化和终末期肝病。目前，肝活检仍然是诊断NASH和确定疾病严重程度的唯一可靠方法。目前临床上可用的非侵入性测试缺乏准确性和可靠性。鉴于NAFLD患病率的急剧增加以及针对NASH患者开发新疗法的重大研究努力，迫切需要非侵入性、简单、可重复和可靠的基于机制的生物标志物，其不仅可以帮助诊断NASH，而且可以作为评估治疗反应和预后的有用终点。因此，该提案的总体目标是定义导致肝细胞死亡的分子机制以及与NAFLD肝损伤和疾病进展的联系，并为患有这种疾病的患者建立基于机制的生物标志物。基于广泛的初步数据，我们提出了一个新的中心假设，即在NAFLD中，肝细胞caspase-3活化在肝损伤的发病机制中起着机械作用，血浆caspase-裂解的细胞角蛋白（CK）-18片段水平是这种疾病患者的理想生物标志物。来自非酒精性脂肪性肝炎NIH临床研究网络（NASH CRN），克利夫兰诊所肥胖和代谢研究所和酒精性脂肪性肝炎病例（CASH）登记处的合作和支持以及半胱天冬酶3基因缺陷小鼠的可用性将成为这些研究的关键工具。本提案是对项目公告PA-07-052“疾病生物标志物的开发”的回应。我们的提案具有以下具体目的：首先，我们将确定非侵入性定量血液中半胱天冬酶产生的CK-18片段水平用于NASH诊断、监测疾病进展和随时间对治疗干预的反应的效用。其次，我们将通过使用人体样本以及NAFLD的体内饮食模型和脂质过载的体外细胞模型，建立肝细胞中半胱天冬酶激活和CK-18裂解与血浆CK-18片段水平增加和疾病进展的时间和因果关系。该提案在技术上和概念上都是创新的，因为它使用复杂的技术测试了脂质诱导肝毒性的新概念。此外，该提案的结果不仅可以为NAFLD疾病进展的具体机制带来新的见解，而且还可以转化为临床上可用的第一个可靠的非侵入性生物标志物，对这种高度常见和潜在严重疾病的患者护理的几个方面产生巨大的积极影响。非酒精性脂肪肝（NAFLD）是一种严重的公共卫生问题。现在估计影响美国30%的成年人和10%的儿童NAFLD代表广泛的疾病，从脂肪肝（通常遵循良性非进展性临床过程）到脂肪性肝炎或NASH（一种更严重的NAFLD形式，可能进展为肝硬化和终末期肝病）。目前，侵入性肝活检仍然是诊断NASH和确定疾病严重程度的唯一可靠方法。我们的初步研究表明，肝活检可能是可以避免的，因为开发了一种新的生物标志物，可以使用简单的血液样本在疑似NAFLD患者中区分NASH和脂肪肝。该生物标志物基于半胱天冬酶3产生的细胞角蛋白18（CK-18）片段，与脂肪肝相比，其在NASH中升高。该提议的结果不仅可以为NAFLD疾病进展的具体机制带来新的见解，从而提出新的治疗策略，而且还可以在相对短期内开发和验证第一个临床可用的、可靠的、非侵入性NAFLD生物标志物。这种生物标志物在这种高度常见和潜在严重疾病的患者护理中可能产生的潜在影响是巨大的。

DESCRIPTION (provided by applicant): Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease worldwide. It is now estimated to affect close to 30% of the adult US population. NAFLD represents a wide spectrum of conditions ranging from simple fatty liver which in general follows a benign non progressive clinical course, to steatohepatitis or NASH, a more serious form of NAFLD characterized by hepatocellular apoptosis, liver injury and inflammation that may progress to cirrhosis and end-stage liver disease. At present time, a liver biopsy remains the only reliable way to diagnose NASH and establish the severity of disease. Current non-invasive clinically available tests lack accuracy and reliability. In light of the dramatic increase in the prevalence of NAFLD in conjunction with the significant research effort in developing novel therapies targeted to those patients with NASH, non invasive, simple, reproducible and reliable mechanism-based biomarkers which can not only help in the diagnosis of NASH, but also be useful endpoints for assessment of treatment response and prognosis are urgently needed. Thus, the overall objectives of this proposal are to define the molecular mechanisms contributing to liver cell death and the link to liver damage and disease progression in NAFLD, as well as to establish a mechanism-based biomarker for patients with this condition. Based on extensive preliminary data, we propose the novel central hypothesis that in NAFLD, hepatocyte caspase-3 activation plays a mechanistic role in the pathogenesis of liver damage and plasma caspase-cleaved Cytokeratin (CK)-18 fragment levels is an ideal biomarker for patients with this condition. The established collaboration and support from the Nonalcoholic Steatohepatitis NIH Clinical Research Network (NASH CRN), the Bariatric and Metabolic Institute at the Cleveland Clinic, and the Case Alcoholic Steatohepatitis (CASH) Registry as well as the availability of caspase 3 genetically deficient mice will serve as critical tools for these studies. This proposal is in response to Program Announcement PA-07-052 "Development of Disease Biomarkers". Our proposal has the following Specific Aims; First, we will determine the utility for non-invasive quantification of caspase-generated CK-18 fragment levels in blood for NASH diagnosis, monitoring disease progression and response to therapeutic interventions over time. Second, we will establish the temporal and causal relationship of caspase activation and CK-18 cleavage in hepatocytes, with increase in plasma CK-18 fragment levels, and disease progression by using human samples as well as in vivo dietary models of NAFLD and in vitro cell models of lipid overloading. The proposal is innovative technically and conceptually as it tests new concepts for lipid induced hepatotoxicity using sophisticated technologies. Moreover, The results of this proposal may not only bring new insights to the specific mechanisms responsible for disease progression in NAFLD, but also could translate into the first reliable noninvasive biomarker clinically available with a tremendous positive impact in several aspects of the care of patients with this highly common and potentially serious condition. PUBLIC HEALTH RELEVANCE: Nonalcoholic fatty liver disease (NAFLD) is a serious public health problem. It is now estimated to affect 30% of adults and 10% of children in the U.S. NAFLD represents a wide spectrum of conditions ranging from fatty liver which in general follows a benign non progressive clinical course, to steatohepatitis or NASH, a more serious form of NAFLD that may progress to cirrhosis and end-stage liver disease. At present time, an invasive liver biopsy remains the only reliable way to diagnose NASH and establish the severity of disease. Our preliminary studies suggest that liver biopsy may be avoidable with the development of a new biomarker that can be used to differentiate NASH from fatty liver in patients with suspected NAFLD, using a simple blood sample. The biomarker is based on caspase 3-generated Cytokeratin 18 (CK-18) fragments, which are elevated in NASH compared with fatty liver. The results of this proposal may not only bring new insights to the specific mechanisms responsible for disease progression in NAFLD, and thus suggesting novel therapeutic strategies, but also may result in a relatively short term, in the development and validation of the first clinically available, reliable, non- invasive NAFLD biomarker. The potential impact that such a biomarker could have in the care of patients for this highly common and potentially serious condition is great.