Identification of the genetic regulators of Plasminogen Activator Inhibitor-1

纤溶酶原激活剂抑制剂-1 遗传调节因子的鉴定

• 批准号: 9171539
• 负责人: Randal J Westrick
• 金额: $ 43.35万
• 依托单位: OAKLAND UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-25 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9171539
• 关键词: Adipose tissue; Affect; Alpha Granule; Antigens; Area; Arteries; Blood Platelets; Blood coagulation; CRISPR/Cas technology; Cardiovascular Diseases; Chromosomes, Human, Pair 5; DNA; Development; Disease; Endothelial Cells; Endothelial Plasminogen Activator Inhibitors; Fibrinolysis; Gene Expression; Genes; Genetic; Genetic Transcription; Genotype; Heart; Hemorrhage; Heterogeneity; Inbred Strains Mice; Individual; Investigation; Liver; Measures; Messenger RNA; Morbidity - disease rate; Mouse Strains; Mus; Mutant Strains Mice; Mutation; Myocardial Infarction; Names; Nucleic Acid Regulatory Sequences; Nucleotide Mapping; One-Step dentin bonding system; Plasma; Plasminogen Activator Inhibitor 1; Process; Production; Quantitative Trait Loci; Regulation; Source; Testing; Thrombosis; Thrombus; Tissues; Transcriptional Regulation; Validation; Variant; Veins; Venous Thrombosis; Work; abstracting; cell type; comparative; genetic variant; genome editing; inhibitor/antagonist; insight; mortality; mouse model; new therapeutic target; prevent; transcriptome; transcriptome sequencing; transcriptomics

Abstract Thrombosis (intravascular blood clots) is a major cause of morbidity and mortality in most areas of the world. Thrombus formation occurs in both arteries and veins and is a dynamic process. Both the development (blood coagulation) and dissolution (fibrinolysis) of thrombi must be exquisitely controlled to prevent either severe bleeding or thrombosis. Elevated levels of an important fibrinolysis inhibitor, plasminogen activator inhibitor-1 (PAI-1, gene name: Serpine1) in plasma have been associated with both venous thrombosis and myocardial infarction. Intra-individual variations in platelet PAI-1 levels could have a major impact on thrombotic disease. We have previously found that an occlusive arterial thrombus could not be formed in a mouse model with complete PAI-1 deficiency. Over 90% of circulating PAI-1 is located in the platelet alpha granules. We measured platelet PAI-1 antigen levels among 12 inbred mouse strains. The C57BL/6J mouse strain has essentially no platelet PAI-1, while the LEWES/EiJ strain has ~100 fold higher platelet PAI-1 than the C57BL/6J strain. We used crosses of the LEWES/EiJ and C57BL/6J strains to generate 24 genetically informative F2 progeny and through QTL analysis identified a major locus for the regulation of platelet PAI-1 levels. This 14.4 megabase region on chromosome 5 contains the PAI-1 gene. We hypothesize that a cis-regulatory expression variant is responsible for controlling platelet PAI-1 expression (pPAI1High). In the studies below, we propose to identify this expression variant through the analysis of additional F2 mice and the production of genome- edited mice for validation of the identified candidate variant(s). We will also determine whether this expression variant affects platelet specific genes other than PAI-1 by analyzing the platelet transcriptomes of LEWES/EiJ and C57BL/6J mice. We have also determined that there are differences in PAI-1 expression and antigen levels in liver, heart and adipose tissue in LEWES/EiJ compared to C57BL/6J. These differences could be due to the same or different expression variants as those controlling platelet PAI-1. Our hypothesis is that endothelial PAI-1 expression is the major source of PAI- 1 from these tissues. The heterogeneity of cell types and relative fraction of endothelial cells in each tissue could have a major impact on PAI-1 levels in these tissues, compromising our ability to draw conclusions regarding the cellular origin of PAI-1. In order to dissect the cell types responsible for these expression differences, we will use a Ribotagged mouse model to specifically pull out endothelial messages from LEWES/EiJ and C57BL/6J and perform a comparative transcriptomic analyses. The proposed investigations of PAI-1 regulation will provide insights into platelet and endothelial specific gene expression as well as providing novel therapeutic targets for modulating thrombosis and fibrinolysis.

抽象的 血栓形成（血管内血栓）是大多数地区发病和死亡的主要原因 世界。血栓形成发生在动脉和静脉中，并且是一个动态过程。两者都 必须严格控制血栓的形成（血液凝固）和溶解（纤维蛋白溶解） 防止严重出血或血栓形成。一种重要的纤维蛋白溶解抑制剂的水平升高， 血浆中的纤溶酶原激活剂抑制剂-1（PAI-1，基因名称：Serpine1）与 静脉血栓和心肌梗塞。血小板 PAI-1 水平的个体差异可能 对血栓性疾病有重要影响。我们之前发现，闭塞的动脉 PAI-1完全缺乏的小鼠模型中不能形成血栓。流通量90%以上 PAI-1 位于血小板α颗粒中。我们测量了 12 名患者的血小板 PAI-1 抗原水平 近交系小鼠品系。 C57BL/6J 小鼠品系基本上没有血小板 PAI-1，而 LEWES/EiJ 菌株的血小板 PAI-1 比 C57BL/6J 菌株高约 100 倍。我们使用了十字架 LEWES/EiJ 和 C57BL/6J 菌株通过 QTL 产生 24 个遗传信息丰富的 F2 后代 分析确定了血小板 PAI-1 水平调节的主要位点。这个 14.4 兆基地区域 5 号染色体含有 PAI-1 基因。我们假设顺式调节表达变体是 负责控制血小板 PAI-1 表达 (pPAI1High)。在下面的研究中，我们建议 通过分析额外的 F2 小鼠和基因组的产生来识别这种表达变体 编辑小鼠以验证已识别的候选变体。我们还将确定这是否 通过分析血小板，表达变异影响除 PAI-1 之外的血小板特异性基因 LEWES/EiJ 和 C57BL/6J 小鼠的转录组。我们还确定存在差异 LEWES/EiJ 中肝脏、心脏和脂肪组织中 PAI-1 表达和抗原水平与 C57BL/6J。这些差异可能是由于相同或不同的表达变体造成的 控制血小板PAI-1。我们的假设是内皮 PAI-1 表达是 PAI-1 的主要来源 1 来自这些组织。细胞类型的异质性和每个细胞中内皮细胞的相对分数 组织可能会对这些组织中的 PAI-1 水平产生重大影响，从而损害我们的绘制能力 关于 PAI-1 细胞起源的结论。为了剖析造成这些的细胞类型 表达差异，我们将使用 Ribotagged 小鼠模型来专门提取内皮细胞 来自 LEWES/EiJ 和 C57BL/6J 的消息并进行比较转录组分析。这 拟议的 PAI-1 调节研究将提供对血小板和内皮特异性的见解 基因表达以及为调节血栓形成和提供新的治疗靶点 纤维蛋白溶解。