Novel proteolytic mechanisms driving pathologic hepatic congestion in drug-induced hepatotoxicity

药物引起的肝毒性中驱动病理性肝充血的新蛋白水解机制

基本信息

批准号：
10638320
负责人：
Bryan L Copple
金额：
$ 49.79万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-11 至 2027-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10638320
关键词：
Acetaminophen Acetylcysteine Active Sites Acute Liver Failure Agonist Analgesics Angiostatins Anti-Inflammatory Agents Automobile Driving Biochemistry Biological Markers Blood Vessels Blood coagulation Breeding Cell Separation Cells Complement Cytolysis Data Dose Elastases Enzyme Precursors Enzymes Equation Failure Fibrinolysis Flow Cytometry Generations Goals Hemorrhage Hepatic Hepatocyte Hepatotoxicity Hospital Costs Human Knockout Mice Leukocytes Life Link Liver Liver Dysfunction Liver Failure Liver diseases MME gene Macrophage Maps Mediating Modality Molecular Morbidity - disease rate Mus Mutation Nature Outcome Overdose Pathologic Pathology Pathway interactions Patients Peripheral Pharmaceutical Preparations Plasma Plasmin Plasminogen Prevention Production Proteins Proteolysis Proteomics Publishing Recommendation Recovery Regulation Research Risk Role Serine Protease Signal Pathway Site Small Interfering RNA System Testing Text Therapeutic Time United States Update Validation Vascular Diseases acetaminophen overdose acetaminophen-induced liver injury acute liver injury axl receptor tyrosine kinase cell type design drug induced liver injury experimental study extracellular genetic approach hepatoprotective hepatotoxin improved outcome inhibitor intrahepatic knock-down lipid nanoparticle liver injury liver repair mortality novel novel therapeutics pharmacologic prevent programs protective effect response thrombolysis vascular injury

项目摘要

Modified Project Summary/Abstract Section The summary/abstract did not require alteration The leading cause of acute liver injury and acute liver failure in the United States is hepatotoxicity caused by overdose of the pain medication acetaminophen (APAP). Even with available treatments (i.e., N-acetylcysteine), more than half of APAP overdose patients require costly hospitalization and antiquated standards to guide treatment. APAP overdose patients would benefit most from treatments that prevent the progression of simple hepatotoxicity to liver failure. Using robust experimental settings and results from acute liver failure patients, our team uncovered a novel pathway whereby macrophage elastase (MMP-12) prevents progression of hepatotoxicity to ALF by reducing excess production of the fibrinolytic enzyme plasmin. The current view is that plasmin production in the APAP-injured liver is controlled by standard proteins involved in blood clot lysis. However, our strong preliminary studies suggest that, in contrast to the current paradigm, hepatic plasmin generation is controlled locally by direct MMP-12-dependent proteolysis of the plasminogen zymogen. The central hypothesis framing these studies is macrophage elastase (MMP-12) prevents vascular damage and intrahepatic congestion after APAP overdose by cleaving plasminogen at a site that releases the anti-inflammatory factor angiostatin while preventing formation of active plasmin. Our approach includes genetically-modified mice with deficiency in components of the signaling pathway controlling MMP-12 expression, mice expressing plasminogen with an active site mutation that prevents plasmin activity, novel lipid nanoparticles to superimpose plasminogen deficiency in experimental APAP hepatotoxicity, and leading edge N-degradomics to pinpoint plasmin substrates in the injured liver. The established investigative team comprises experts in APAP-induced liver injury and repair, biochemistry/function of the fibrinolytic system, vascular pathology, leading-edge proteomic analysis, and experimental settings of liver disease. In our proposed studies we will: define the cellular and molecular mechanisms driving MMP-12 expression after acetaminophen overdose (Aim 1); determine the mechanisms whereby MMP-12 prevents pathologic vascular congestion in the acetaminophen-injured liver (Aim 2); and determine the role of plasmin(ogen) proteolytic and non-proteolytic functions in APAP-induced liver injury (Aim 3). The expected outcome of these Specific Aims is the discovery of a novel mechanistic link between leukocyte effector function (i.e., MMP-12 expression) and prevention of intrahepatic hyperfibrinolysis in the injured liver, including evidence of a novel molecular mechanism controlling plasmin generation. This could pinpoint mechanistic biomarkers to predict imminent hepatic vascular dysfunction in the injured liver and to uncover therapeutic modalities to prevent hepatic congestion after hepatotoxicant exposure. By virtue of separation from traditional regulation of fibrinolysis, this could greatly improve outcome in APAP overdose without increasing bleeding risk.

修改后的项目摘要/摘要部分摘要/摘要不需要修改在美国，急性肝损伤和急性肝功能衰竭的主要原因是过量服用止痛药对乙酰氨基酚 (APAP) 引起的肝毒性。即使有可用的治疗方法（即 N-乙酰半胱氨酸），超过一半的 APAP 过量患者也需要昂贵的住院费用和过时的标准来指导治疗。 APAP 过量患者将从预防单纯肝毒性进展为肝衰竭的治疗中获益最多。利用稳健的实验设置和急性肝衰竭患者的结果，我们的团队发现了一种新的途径，巨噬细胞弹性蛋白酶 (MMP-12) 通过减少纤溶酶纤溶酶的过量产生来防止 ALF 肝毒性的进展。目前的观点是，APAP 损伤的肝脏中纤溶酶的产生是由参与血凝块溶解的标准蛋白质控制的。然而，我们强有力的初步研究表明，与目前的范式相反，肝脏纤溶酶的生成是通过纤溶酶原酶原的直接 MMP-12 依赖性蛋白水解来局部控制的。这些研究的核心假设是巨噬细胞弹性蛋白酶（MMP-12）通过在释放抗炎因子血管抑制素的位点裂解纤溶酶原，同时防止活性纤溶酶的形成，从而防止 APAP 过量后的血管损伤和肝内充血。我们的方法包括控制 MMP-12 表达的信号通路成分缺陷的转基因小鼠、表达具有阻止纤溶酶活性的活性位点突变的纤溶酶原的小鼠、在实验性 APAP 肝毒性中叠加纤溶酶原缺陷的新型脂质纳米颗粒，以及前沿 N -降解组学以查明受损肝脏中的纤溶酶底物。已成立的研究团队由 APAP 诱导的肝损伤和修复、生物化学/纤溶系统功能、血管病理学、前沿蛋白质组学分析和肝病实验设置方面的专家组成。在我们提出的研究中，我们将：定义对乙酰氨基酚过量后驱动 MMP-12 表达的细胞和分子机制（目标 1）；确定 MMP-12 预防对乙酰氨基酚损伤肝脏中病理性血管充血的机制（目标 2）；并确定纤溶酶（原）蛋白水解和非蛋白水解功能在 APAP 诱导的肝损伤中的作用（目标 3）。这些具体目标的预期结果是发现白细胞效应功能（即 MMP-12 表达）与预防受损肝脏中肝内纤维蛋白溶解亢进之间的新机制联系，包括控制纤溶酶生成的新分子机制的证据。这可以精确定位机械生物标志物，以预测受损肝脏中即将发生的肝血管功能障碍，并揭示预防肝毒物暴露后肝充血的治疗方式。由于与传统的纤溶调节分离，这可以极大地改善 APAP 过量的结果，而不会增加出血风险。