Molecular mechanism of senile cardiac amyloidosis

老年心脏淀粉样变的分子机制

• 批准号: 9558425
• 负责人: Lawreen H. Connors
• 金额: $ 41.15万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9558425
• 关键词: Age of Onset; Aged, 80 and over; American; Amyloid; Amyloidosis; Area; Attention; Binding; Binding Sites; Biochemical; Biological Markers; Boston; Brain natriuretic peptide; Cardiac; Cardiac Myocytes; Cardiopulmonary; Cells; Cellular Stress; Clinical; Clinical Research; Cohort Studies; Complex; Controlled Study; Coupled; Data; Deposition; Detection; Diagnosis; Diflunisal; Disease; EFRAC; Elderly; Enrollment; Enzyme-Linked Immunosorbent Assay; Etiology; Evaluation; Genetic; Half-Life; Heart failure; Hepatocyte; Immunoblot Analysis; International; Investigation; Left; Left Ventricular Ejection Fraction; Life; Link; Location; Longitudinal prospective study; MMP9 gene; Mass Spectrum Analysis; Measures; Messenger RNA; Modeling; Molecular; Molecular Chaperones; Molecular Weight; Morbidity - disease rate; Nature; Nucleic Acid Regulatory Sequences; Patients; Peptides; Persons; Pharmaceutical Preparations; Population; Prealbumin; Prevalence; Process; Proteins; Regulator Genes; Reporting; Research; Retinol Binding Proteins; Risk; Risk Factors; Role; Route; Sampling; Series; Serological; Serum; Single Nucleotide Polymorphism; Spectrum Analysis; Staining method; Stains; Structural Protein; Structure; Surface Plasmon Resonance; Testing; Thick; Tissues; United States National Institutes of Health; Universities; Untranslated RNA; Uric Acid; Ventricular; aging population; amyloid fibril formation; base; cell type; cohort; effective intervention; experience; follow-up; genetic variant; glycosylation; heme oxygenase-1; induced pluripotent stem cell; modifiable risk; monocyte; mortality; outcome forecast; polypeptide C; protein expression; protein structure; proteotoxicity; response; stoichiometry; sulfated glycoprotein 2; survival prediction; translational approach

ABSTRACT Heart failure caused by wild-type transthyretin amyloidosis (ATTRwt) is an underappreciated cause of morbidity and mortality in the aging population. With past support from NIH/NIA (R01AG031804) for studies of ATTRwt, we have characterized the biochemical nature of wild-type TTR in sera and tissues, uncovered a role for clusterin (CLU), identified disease-associated non-coding TTR genetic variants, and accurately defined clinical measures in a large series to identify predictors of survival. Based on these data, we now hypothesize that the pathobiology of ATTRwt involves protein and genetic modifiers, i.e. ATTRwt amyloid fibril formation is related to aberrant or disrupted interactions between TTR and binding partners, clusterin (CLU) and retinol binding protein (RBP4), and the presence of single nucleotide polymorphisms (SNPs) in TTR gene regulatory regions underlie the process. To test our hypothesis, we propose three integrated and translational aims: SA 1. To clarify the amyloidogenic nature of wild-type TTR by interrogating TTR binding partners and SNPs identified in patient samples. Our plan is to a) characterize the structures of CLU and RBP4 isolated from patient and control sera by mass spectrometry (MS), b) define TTR:CLU molecular interactions using MS, surface plasmon resonance, and spectroscopy with attention to CLU glycosylation, stoichiometric effects, and binding site locations, c) study CLU interactions with complexed TTR-RBP4 as in SA1b, and d) correlate TTR SNPs (rs72922940, rs3794885, rs3764479) to CLU and RBP4 structural data from SA1a. SA2. To delineate the roles of TTR SNPs, CLU and RBP4, and the impact of diflunisal (TTR tetramer stabilizing drug) in ATTRwt using a patient-derived, cell-based model. Induced pluripotent stem cells (iPSC), reprogrammed from ATTRwt and control blood monocytes and differentiated into hepatocytes (TTR, CLU, RBP4 expression cell type) or cardiomyocytes (TTR amyloid target cell type) will be used to a) link TTR SNPs to TTR expression/secretion by comparing TTR mRNA half-life/concentrations and protein levels (intracellular and secreted) in ATTRwt hepatocytes +/- SNPs from SA1d; b) define molecular associations of TTR, CLU, and RBP4 secreted by ATTRwt and control hepatocytes using MS and PAGE; and c) compare ATTRwt cardiomyocyte response to ATTRwt hepatocyte-secreted TTR and binding partners +/- diflunisal (and +/- CLU peptides from SA1b) by measuring expression of cardiac cell stress markers (MMP9, HO1, Hsp27, p21, cTnT, BNP) implicated in amyloid pathobiology using qPCR and immunoblot analyses. SA3. To examine associations of TTR, CLU, RBP4, and SNPs with progression and survival in ATTRwt, and the impact of diflunisal on disease course. We will a) measure serum levels of TTR, CLU, and RBP4 in patients at baseline and follow-up evaluations by ELISA, b) correlate serological and genetic data to ATTRwt survival risk factors (BNP, UA, RWT and LVEF), and c) quantify the effect of diflunisal in an observational, controlled study of ATTRwt by assessing investigational and survival risk factors at baseline and follow-up.

抽象的 野生型运甲状腺素蛋白淀粉样变性 (ATTRwt) 引起的心力衰竭是一个未被充分认识的原因 人口老龄化的发病率和死亡率。在 NIH/NIA (R01AG031804) 过去的支持下进行的研究 ATTRwt，我们表征了血清和组织中野生型 TTR 的生化性质，发现了其作用 针对 clusterin (CLU)，识别出与疾病相关的非编码 TTR 遗传变异，并准确定义 一系列的临床测量以确定生存的预测因素。根据这些数据，我们现在假设 ATTRwt 的病理学涉及蛋白质和遗传修饰剂，即 ATTRwt 淀粉样原纤维的形成是 与 TTR 和结合伙伴、簇蛋白 (CLU) 和视黄醇之间的异常或破坏的相互作用有关 结合蛋白 (RBP4) 以及 TTR 基因调控中单核苷酸多态性 (SNP) 的存在 区域是这一过程的基础。为了检验我们的假设，我们提出了三个综合和转化目标： SA 1. 通过询问 TTR 结合伴侣来阐明野生型 TTR 的淀粉样蛋白生成性质 在患者样本中鉴定出的 SNP。我们的计划是 a) 表征分离的 CLU 和 RBP4 的结构 通过质谱 (MS) 从患者和对照血清中提取，b) 使用 MS 定义 TTR:CLU 分子相互作用， 表面等离子体共振和光谱学，关注 CLU 糖基化、化学计量效应和 结合位点位置，c) 研究 CLU 与 SA1b 中的复合 TTR-RBP4 相互作用，以及 d) 关联 TTR SA1a 中 CLU 和 RBP4 结构数据的 SNP（rs72922940、rs3794885、rs3764479）。 SA2。描述 TTR SNP、CLU 和 RBP4 的作用以及二氟尼柳（TTR 四聚体）的影响 ATTRwt 中使用源自患者的细胞模型。诱导多能干细胞 (iPSC)，从 ATTRwt 和对照血单核细胞重编程并分化为肝细胞 (TTR、 CLU、RBP4 表达细胞类型）或心肌细胞（TTR 淀粉样蛋白靶细胞类型）将用于 a) 连接 TTR 通过比较 TTR mRNA 半衰期/浓度和蛋白质水平来确定 TTR 表达/分泌的 SNP ATTRwt 肝细胞中的（细胞内和分泌的）+/- SA1d 的 SNP； b) 定义分子关联 使用 MS 和 PAGE 检测 ATTRwt 和对照肝细胞分泌的 TTR、CLU 和 RBP4； c) 比较 ATTRwt 心肌细胞对 ATTRwt 肝细胞分泌的 TTR 和结合伴侣 +/- 二氟尼柳（和 +/- SA1b 的 CLU 肽），通过测量心肌细胞应激标记物（MMP9、HO1、Hsp27、 使用 qPCR 和免疫印迹分析发现 p21、cTnT、BNP）与淀粉样蛋白病理学有关。 SA3。为了检查 TTR、CLU、RBP4 和 SNP 与 ATTRwt 进展和生存的关联， 以及二氟尼柳对病程的影响。我们将 a) 测量 TTR、CLU 和 RBP4 的血清水平 通过 ELISA 对患者进行基线和后续评估，b) 将血清学和遗传数据与 ATTRwt 相关联 生存风险因素（BNP、UA、RWT 和 LVEF），以及 c) 量化二氟尼柳在观察性、 通过评估基线和随访时的研究和生存风险因素来进行 ATTRwt 对照研究。