Discovery of Novel Interventions of the Antiphospholipid Syndrome

抗磷脂综合征新干预措施的发现

基本信息

批准号：
8163148
负责人：
Chieko Mineo
金额：
$ 39.63万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8163148
关键词：
Adaptor Signaling Protein Address Adhesions Anticoagulants Antiphospholipid Antibodies Antiphospholipid Syndrome Autoimmune Diseases Binding Biological Assay Biological Availability Blocking Antibodies Blood Platelets Blood Vessels Bone Marrow Cadherins Cardiovascular Diseases Cardiovascular system Cell Culture Techniques Cell Surface Receptors Cells Coagulation Process Cultured Cells Development Disabled Persons Disease Endothelial Cells Endothelium Enhancing Antibodies Enzymes Functional disorder Genetic Goals Inflammation Intervention Knock-in Mouse Knowledge Leukocytes Mediating Medical Research Molecular Molsidomine Mus Myocardial Infarction Neurons Nitric Oxide Outcome Pathogenesis Patients Phenotype Phosphoric Monoester Hydrolases Phosphorylation Process Protein Dephosphorylation Protein Phosphatase 2A Regulatory Subunit PR53 RNA Interference Research Research Project Grants Risk Role Series Serine Signal Transduction Signaling Molecule Stroke Surface Testing Therapeutic Thrombosis Thrombus Translating Treatment Efficacy United States National Institutes of Health Vascular Diseases Wild Type Mouse Work antibody engineering apolipoprotein E receptor 2 attenuation base cadherin 5 combat efficacy testing human NOS3 protein in vivo intravital microscopy loss of function meetings monocyte mouse model mutant new therapeutic target novel novel therapeutics prevent promoter prophylactic recombinase reconstitution research study response small molecule

项目摘要

DESCRIPTION (provided by applicant): The antiphospholipid syndrome (APS) is an autoimmune disease characterized by a markedly increased risk of thromboses and cardiovascular diseases resulting from elevated levels of circulating antiphospholipid antibodies (aPL). Alterations in the function of vascular cells induced by aPL underlie these outcomes; however, the molecular basis of aPL action is not clear. Our recently completed studies indicate that aPL isolated from APS patients fully antagonize endothelial nitric oxide synthase (eNOS) through impaired phosphorylation of the enzyme at S1177 via the phosphatase PP2A. aPL inhibition of eNOS results in increases in endothelial-leukocyte adhesion and thrombosis. We have also discovered that the cell surface receptor, apolipoprotein E receptor 2 (apoER2) is required for induction of vascular dysfunction by aPL. The overall goals of the proposed research are first to determine the molecular basis of aPL-apoER2 actions and second to test novel interventions directed at the mechanism. Aim 1 will determine how aPL-apoER2 induces vascular dysfunction. The requirement for the adaptor molecule Dab-1 will be tested in cultured endothelial cells and isolated platelets using loss-of-function strategy. Using intravital microscopy, the in vivo role of Dab-1 in aPL-induced leukocyte adhesion and thrombus formation will be assessed in wild-type vs. knock-in mice that express mutant apoER2 incapable of interacting with Dab-1. We will also determine how aPL activates PP2A. Our focus will be to identify and characterize the regulatory B subunits of PP2A required for aPL-mediated eNOS antagonism. Furthermore, we will test if the in vivo effect of aPL on leukocyte adhesion and thrombus formation are mediated by impaired eNOS S1177 phosphorylation by using the S1177D eNOS knock-in mouse in which eNOS is constitutively-active and not amenable to dephosphorylation by PP2A. Aim 2 will determine the critical aPL target cells in vivo. The bone marrow reconstitution between apoER2+/+ and apoER2-/- mice will be used to assess the role of bone-marrow derived platelets and monocytes. The specific role of endothelium will be tested by crossing floxed apoER2 mice with mice expressing Cre-recombinase regulated by endothelial cadherin promoter to delete apoER2 from endothelium. In both studies, we will use intravital microscopy to assay for aPL-induced leukocyte adhesion and thrombosis. Aim 3 will test if novel interventions directed at apoER2- and eNOS- related mechanisms prevent the aPL-induced leukocyte adhesion and thrombosis. Experiments will explore whether an engineered antibody that promotes clearance of circulating aPL will prevent aPL-induced vascular responses. A blocking antibody or a small molecule that interferes aPL interaction with target cells will also be tested. The impact of provision of exogenous NO by Molsidomine treatment will also be assessed. Leukocyte adhesion and thrombosis induced by aPL will be assayed by intravital microscopy in wild-type mice. Together these aims will determine how aPL cause leukocyte adhesion and thrombosis and test potential therapeutic strategies to prevent aPL-mediated vascular dysfunction. PUBLIC HEALTH RELEVANCE: The goals of the proposed research project are to elucidate the molecular basis of APS using cultured cells and a series of complementary mouse models and to further test novel interventions directly based on these pathogenic mechanisms. We anticipate that the new knowledge gained will then be rapidly translated into new prophylactic or therapeutic strategies to combat the devastating impact of APS. Considering the staggering impact of vascular diseases on the patients suffering from APS, the goals of the research project are in line with the objectives of the National Institute of Health to address these disorders through medical research.

描述（由申请人提供）：抗磷脂综合征（APS）是一种自身免疫性疾病，其特征是由于循环抗磷脂抗体（aPL）水平升高而导致血栓形成和心血管疾病的风险显着增加。 aPL 引起的血管细胞功能的改变是这些结果的基础；然而，aPL 作用的分子基础尚不清楚。我们最近完成的研究表明，从 APS 患者中分离出的 aPL 通过磷酸酶 PP2A 损害内皮型一氧化氮合酶 (eNOS) S1177 的磷酸化，从而完全拮抗内皮型一氧化氮合酶 (eNOS)。 aPL 抑制 eNOS 会导致内皮-白细胞粘附和血栓形成增加。我们还发现，细胞表面受体载脂蛋白 E 受体 2 (apoER2) 是 aPL 诱导血管功能障碍所必需的。拟议研究的总体目标首先是确定 aPL-apoER2 作用的分子基础，其次是测试针对该机制的新干预措施。目标 1 将确定 aPL-apoER2 如何诱导血管功能障碍。将使用功能丧失策略在培养的内皮细胞和分离的血小板中测试对接头分子 Dab-1 的需求。使用活体显微镜，将在表达不能与 Dab-1 相互作用的突变 apoER2 的野生型小鼠和敲入小鼠中评估 Dab-1 在 aPL 诱导的白细胞粘附和血栓形成中的体内作用。我们还将确定 aPL 如何激活 PP2A。我们的重点是鉴定和表征 aPL 介导的 eNOS 拮抗作用所需的 PP2A 调节 B 亚基。此外，我们将使用 S1177D eNOS 敲入小鼠测试 aPL 对白细胞粘附和血栓形成的体内作用是否是由 eNOS S1177 磷酸化受损介导的，其中 eNOS 具有组成型活性，并且不易于被 PP2A 去磷酸化。目标 2 将确定体内关键的 aPL 靶细胞。 apoER2+/+ 和 apoER2-/- 小鼠之间的骨髓重建将用于评估骨髓来源的血小板和单核细胞的作用。通过将 floxed apoER2 小鼠与表达由内皮钙粘蛋白启动子调节的 Cre 重组酶的小鼠杂交以从内皮中删除 apoER2 来测试内皮的具体作用。在这两项研究中，我们将使用活体显微镜来检测 aPL 诱导的白细胞粘附和血栓形成。目标 3 将测试针对 apoER2 和 eNOS 相关机制的新干预措施是否可以预防 aPL 诱导的白细胞粘附和血栓形成。实验将探讨促进循环 aPL 清除的工程化抗体是否会阻止 aPL 诱导的血管反应。还将测试干扰 aPL 与靶细胞相互作用的阻断抗体或小分子。还将评估通过吗西多明治疗提供外源性 NO 的影响。 aPL 诱导的白细胞粘附和血栓形成将通过野生型小鼠的活体显微镜进行测定。这些目标共同将确定 aPL 如何引起白细胞粘附和血栓形成，并测试预防 aPL 介导的血管功能障碍的潜在治疗策略。公共健康相关性：拟议研究项目的目标是利用培养细胞和一系列互补的小鼠模型阐明 APS 的分子基础，并进一步测试直接基于这些致病机制的新干预措施。我们预计获得的新知识将迅速转化为新的预防或治疗策略，以对抗 APS 的破坏性影响。考虑到血管疾病对 APS 患者的巨大影响，该研究项目的目标与美国国立卫生研究院通过医学研究解决这些疾病的目标一致。