Discovery of Novel Interventions of the Antiphospholipid Syndrome

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，其特征是循环抗磷脂抗体（aPL）水平升高导致血栓形成和心血管疾病的风险显著增加。aPL诱导的血管细胞功能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患者分离的aPL完全拮抗内皮型一氧化氮合酶（eNOS）通过受损的磷酸化酶在S1177通过磷酸酶PP 2A。aPL对eNOS的抑制导致内皮-白细胞粘附和血栓形成的增加。我们还发现，细胞表面受体，载脂蛋白E受体2（apoER 2）是由aPL诱导血管功能障碍所必需的。拟议研究的总体目标是首先确定aPL-apoER 2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-apoER 2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衔接子分子Dab-1的需求。使用活体显微镜，在表达不能与Dab-1相互作用的突变体apoER 2的野生型与敲入小鼠中评估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PP 2A。我们的重点将是确定和表征的调控B亚基的PP 2A所需的aPL介导的eNOS拮抗作用。此外，我们将测试aPL对白细胞粘附和血栓形成的体内作用是否是通过使用eNOS S1177 D敲入小鼠的eNOS S1177磷酸化受损介导的，其中eNOS是组成型活性的并且不适合于通过PP 2A去磷酸化。目的2确定体内关键的aPL靶细胞。apoER 2 +/+和apoER 2-/-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。将通过将floxed apoER 2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删除apoER 2来测试内皮的特异性作用。在这两项研究中，我们将使用活体显微镜检测APL诱导的白细胞粘附和血栓形成。目的3将测试针对apoER 2和eNOS相关机制的新干预是否预防aPL诱导的白细胞粘附和血栓形成。实验将探索促进循环aPL清除的工程抗体是否会阻止aPL诱导的血管反应。还将检测阻断抗体或干扰aPL与靶细胞相互作用的小分子。还将评估通过吗西多明治疗提供外源性NO的影响。在野生型小鼠中，通过活体显微镜检查分析aPL诱导的白细胞粘附和血栓形成。这些目标将共同确定aPL如何引起白细胞粘附和血栓形成，并测试潜在的治疗策略，以防止aPL介导的血管功能障碍。

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.