Anti-Coagulant and Cytoprotective activity in CCM pathogenesis

摘要 脑海绵状血管畸形(CCM)是一种急性和慢性出血。 这是该病发病率和死亡率的主要来源。我们发现内皮细胞 在小鼠和人体内，CCM表达显著增加的血栓调节蛋白(TM)和 内皮细胞蛋白C受体(EPCR)，导致内源性抗凝剂的激活 蛋白C。我们假设CCM形成一个抗凝的血管结构域，并被激活 蛋白C(APC)与CCM的出血有关。APC还可发挥细胞保护作用。 通过PAR1信号转导内皮细胞，除其他作用外，内皮细胞稳定 细胞-细胞连接。事实上，APC的这种细胞保护作用已经被创造出来 选择性地维持细胞保护活性的功能突变体的APC丧失。因此，我们 假设APC细胞保护活性可能限制CCM的发病率，就像它在 实验性中风。为了从基因上检验这一假设，我们将检查急性脑出血患者的 F5R504Q/wt因子V Leiden(F5R504Q/wt)小鼠CCM抗凝剂抵抗模型的建立 APC的效果。作为第二种方法，我们将测试单抗MAPC1591的效果 这阻断了急性CCM模型中APC的抗凝活性。测试……的效果 细胞保护活性，我们将检查急性CCM病变的发展和出血 F2rR46Q/R46Q小鼠携带选择性抵抗APC切割的PAR1。相反，我们会 检测3K3A-APC的影响，3K3A-APC是一种功能缺失的APC突变体，在 急、慢性CCM模型的抗凝作用。我们将利用我们的观察结果， 短期低氧或低氧诱导因子1A的药理稳定作用显著 加剧围产期小鼠急性CCM的形成，并使一种强大的亚急性模型成为可能 在成年小鼠身上表现出来。一种既出血又含铁血黄素的亚急性模型 沉积，将用于测试目标1和2中描述的干预措施。完成这些 AIMS将提供对TM和EPCR显著增加的作用的机械性洞察 在CCM中观察到，并提供了关于出血或疾病的重要临床前测试 进展可以通过操纵已经在治疗中的通路来影响 针对中风、败血症和血友病等疾病。

ABSTRACT Cerebral Cavernous Malformations (CCM) are subject to acute and chronic bleeding that is a major source of morbidity and mortality in this disease. We have found that endothelial cells within murine and human CCM express markedly increased levels of thrombomodulin (TM) and endothelial protein C receptor (EPCR), which lead to activation of endogenous anti-coagulant protein C. We hypothesize that CCM form an anti-coagulant vascular domain and that activated Protein C (APC) contributes to bleeding in CCM. APC can also exert a cytoprotective effect on endothelium by signaling via PAR1 resulting in, among other effects, stabilization of endothelial cell-cell junctions. Indeed, this cytoprotective effect of APC has been exploited by creation of APC loss of function mutants that selectively maintain cytoprotective activity. We thus hypothesize that APC cytoprotective activity may limit morbidity from CCM as it does in experimental stroke. To genetically test this hypothesis, we will examine the bleeding in the acute murine CCM models in Factor V Leiden (F5R504Q/wt) mice, which are resistant to the anti-coagulant effect of APC. As a second approach we will test the effect of MAPC1591, a monoclonal antibody that blocks the anti-coagulant activity of APC in acute models of CCM. To test the effects of cytoprotective activity, we will examine acute CCM lesion development and bleeding in F2rR46Q/R46Q mice bearing PAR1 that is selectively resistant to APC cleavage. Conversely, we will examine the effect of 3K3A-APC, a loss of function APC mutant that is selectively impaired in anti-coagulant function in acute and chronic CCM models. We will exploit our observation that a brief period of hypoxia or pharmacological stabilization of Hypoxia-inducible factor 1A markedly exacerbates acute CCM formation in perinatal mice and enables a robust subacute model that manifests in adult mice. This subacute model which manifests both bleeding and hemosiderin deposition, will be used to test the interventions described in aims 1 and 2. Completion of these Aims will provide mechanistic insight into the role of the marked increase of TM and EPCR that is observed in CCM and provide important preclinical tests of the idea that hemorrhage or disease progression can be influenced by manipulating pathways that are already being therapeutically targeted in diseases such as stroke, sepsis, and hemophilia.