Discovery of Novel Interventions of the Antiphospholipid Syndrome

抗磷脂综合征的新干预措施的发现

• 批准号: 8501671
• 负责人: Chieko Mineo
• 金额: $ 37.84万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 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/8501671
• 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; Genetic Engineering; 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

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.

描述(申请人提供)：抗磷脂综合征(APS)是一种自身免疫性疾病，其特征是由于循环中的抗磷脂抗体(APL)水平升高，导致血栓和心血管疾病的风险显著增加。APL诱导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患者中分离的APL通过磷酸酶PP2A使S1177处的内皮型一氧化氮合酶(ENOS)磷酸化受损，从而完全拮抗该酶。APL抑制eNOS导致内皮-白细胞黏附增加和血栓形成。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。这项拟议研究的总体目标是首先确定APL-ApoER2作用的分子基础，其次测试针对该机制的新干预措施。目的1将确定APL-ApoER2如何导致血管功能障碍。对接头分子dab-1的需求将在培养的内皮细胞和分离的血小板中使用功能丧失策略进行测试。利用活体显微镜，将在野生型和转基因小鼠中评估DAB-1在APL诱导的白细胞黏附和血栓形成中的作用，这些小鼠表达突变的ApoER2，而不能与DAB-1相互作用。我们还将确定APL是如何激活PP2A的。我们的重点将是确定和表征APL介导的eNOS拮抗所需的PP2A调节B亚单位。此外，我们将使用eNOS具有结构性活性且不受PP2A去磷酸化影响的S1177D eNOS敲入小鼠，测试APL对白细胞黏附和血栓形成的体内效应是否通过eNOS S1177的磷酸化受损而介导。AIM 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被用来评估骨髓来源的血小板和单核细胞的作用。将ApoER2小鼠与表达受内皮钙粘蛋白启动子调控的Cre重组酶的小鼠杂交，从内皮中删除ApoER2，以测试内皮的特殊作用。在这两项研究中，我们将使用活体显微镜来检测APL诱导的白细胞黏附和血栓形成。目的3将测试针对ApoER2和eNOS相关机制的新干预措施是否可以防止APL诱导的白细胞黏附和血栓形成。实验将探索一种促进循环APL清除的工程抗体是否会阻止APL诱导的血管反应。还将测试干扰APL与靶细胞相互作用的封闭抗体或小分子。还将评估莫西多明治疗提供外源性NO的影响。APL诱导的白细胞黏附和血栓形成将通过活体显微镜在野生型小鼠中进行检测。这些目标将共同决定APL如何导致白细胞黏附和血栓形成，并测试预防APL介导的血管功能障碍的潜在治疗策略。