Caspase Dependent Cell Death in Nonalcoholic Fatty Liver Disease

描述(由申请者提供)：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代表了一系列的疾病，从一般遵循良性非进行性临床病程的单纯性脂肪肝到脂肪性肝炎或NASH，NASH是一种更严重的NAFLD形式，其特征是肝细胞凋亡、肝脏损伤和炎症，可能进展为肝硬化和终末期肝病。目前，肝脏活检仍然是诊断NASH和确定疾病严重程度的唯一可靠的方法。目前临床上可用的非侵入性检测缺乏准确性和可靠性。鉴于NAFLD发病率的急剧上升，以及针对NASH患者开发新的治疗方法的重大研究努力，迫切需要非侵入性、简单、可重复性和可靠的基于机制的生物标志物，这些生物标志物不仅可以帮助NASH的诊断，而且可以作为评估治疗反应和预后的有用终点。因此，这项建议的总体目标是确定导致肝细胞死亡的分子机制以及与NAFLD肝损害和疾病进展的联系，并为这种情况的患者建立一个基于机制的生物标记物。基于大量的初步数据，我们提出了一个新的中心假设，即在NAFLD中，肝细胞caspase-3的激活在肝损伤的发病机制中发挥了作用，而血浆caspase裂解的细胞角蛋白(CK)-18片段水平是这种情况下患者理想的生物标志物。非酒精性脂肪性肝炎NIH临床研究网络(NASH CRN)、克利夫兰诊所的减肥和代谢研究所、酒精性脂肪性肝炎(CASH)病例登记以及caspase 3基因缺陷小鼠的可用性所建立的合作和支持将成为这些研究的关键工具。这项建议是对PA-07-052“疾病生物标记物的发展”计划公告的回应。我们的建议有以下具体目标：首先，我们将确定无创性定量检测血液中caspase产生的CK-18片段水平对NASH诊断、监测疾病进展和随着时间的推移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体内饮食模型和体外细胞脂质过载模型，建立肝细胞caspase激活和CK-18裂解与血浆CK-18片段水平升高和疾病进展的时间和因果关系。该提案在技术和概念上都是创新的，因为它使用尖端技术测试了脂质诱导的肝脏毒性的新概念。此外，这项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，还可能转化为临床上第一个可靠的非侵入性生物标记物，在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。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，在美国，30%的成年人和10%的儿童会受到影响。NAFLD代表了一系列的疾病，从一般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，可能发展为肝硬变和终末期肝病。目前，有创肝活检仍然是诊断NASH和确定疾病严重程度的唯一可靠的方法。我们的初步研究表明，随着一种新的生物标志物的开发，肝脏活检可能是可以避免的，该生物标志物可以使用简单的血液样本来区分可疑NAFLD患者的NASH和脂肪肝。该生物标记物基于caspase 3生成的细胞角蛋白18(CK-18)片段，与脂肪肝相比，NASH患者的CK-18片段水平升高。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，而且可能在相对较短的时间内开发和验证第一个临床可用的、可靠的、非侵入性的NAFLD生物标记物。对于这种非常常见和潜在严重的疾病，这样的生物标记物在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.