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）是世界范围内慢性肝病的主要原因。据估计，目前有近30%的美国成年人患有糖尿病。NAFLD代表了广泛的疾病范围，从单纯性脂肪肝（通常遵循良性非进行性临床病程）到脂肪性肝炎或NASH（一种更严重的NAFLD形式，以肝细胞凋亡、肝损伤和炎症为特征，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。目前临床可用的无创检测缺乏准确性和可靠性。鉴于NAFLD患病率的急剧上升，以及针对NASH患者开发新疗法的重大研究努力，迫切需要无创、简单、可重复和可靠的基于机制的生物标志物，不仅可以帮助NASH的诊断，而且还可以作为评估治疗反应和预后的有用终点。因此，本提案的总体目标是确定NAFLD中导致肝细胞死亡的分子机制以及与肝损伤和疾病进展的联系，并为该疾病患者建立基于机制的生物标志物。基于广泛的初步数据，我们提出了新的中心假设，即在NAFLD中，肝细胞caspase-3激活在肝损伤的发病机制中起着机制作用，血浆caspase-cleaved Cytokeratin (CK)-18片段水平是该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、酒精性脂肪性肝炎病例登记处（CASH）以及caspase 3基因缺陷小鼠的既定合作和支持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”的响应。我们的建议有以下具体目标：首先，我们将确定非侵入性定量血液中caspase产生的CK-18片段水平在NASH诊断、监测疾病进展和对治疗干预的反应方面的效用。其次，我们将通过使用人类样本以及NAFLD的体内饮食模型和体外脂质超载细胞模型，建立肝细胞中caspase激活和CK-18切割的时间和因果关系，血浆CK-18片段水平的增加和疾病进展。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，这一建议的结果不仅可能为NAFLD疾病进展的具体机制带来新的见解，而且还可能转化为临床可用的第一个可靠的无创生物标志物，在这一高度常见和潜在严重疾病患者的护理方面产生巨大的积极影响。公共卫生相关性：非酒精性脂肪性肝病（NAFLD）是一个严重的公共卫生问题。据估计，目前在美国有30%的成人和10%的儿童受到NAFLD的影响。NAFLD代表了广泛的疾病范围，从通常遵循良性非进行性临床病程的脂肪肝到脂肪性肝炎或NASH，这是一种更严重的NAFLD形式，可能发展为肝硬化和终末期肝病。目前，有创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，随着一种新的生物标志物的发展，肝活检可能是可以避免的，这种生物标志物可以通过简单的血液样本来区分疑似NAFLD患者的NASH和脂肪肝。该生物标志物基于caspase 3生成的细胞角蛋白18 （CK-18）片段，与脂肪肝相比，NASH中CK-18片段升高。这一建议的结果不仅可能为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，而且可能在相对短期内开发和验证第一个临床可用的、可靠的、非侵入性的NAFLD生物标志物。这种生物标志物可能对这种非常常见且可能严重的疾病的患者的护理产生巨大的潜在影响。

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.