Molecular and cellular etiology of cerebral cavernous malformations

描述（由申请人提供）：本项目旨在阐明脑海绵状畸形（CCMs）的发病机制，CCMs是一种以静脉窦为特征的脑和脊髓临床综合征，易导致颅内出血。三种基因已被确定为CCMs的病因；KRIT1 （CCM1）、MGC4607 （CCM2）和PDCD10 (CCM3)，这些基因突变后会导致神经血管畸形的形成。我们认为，在血管生成、重塑和/或微血管稳态过程中，CCM基因调控β 1整合素介导的信号通路，而这一过程的扰动有助于CCM的发展。我们在培养的人血管内皮细胞中特异性地沉默了krit1的表达，并观察到细胞增殖、运动和存活的显著扰动。我们还证明了：1)krit1与icap1alpha的相互作用，icap1alpha是一种已知的β a1整合素信号传导介质；2) krit1与MGC4607的相互作用；3) sirna介导的krit1或icap1alpha沉默后，内皮细胞增殖、迁移和存活受到干扰，并分别通过局灶黏附激酶（FAK）、Rac1和整合素连接激酶（ILK）级联反应导致信号减少；4)通过将icap1靶向细胞膜，增强多种β a1整合素信号级联；5) krit1沉默后，icap1alpha和MGC4607的核定位和降解失败；6)有文献记载的Ccm1 （santa, san）和Ccm2 （valentine, vtn）斑马鱼的心血管缺陷；7)san斑马鱼血管内皮细胞凋亡增加导致血管系统紊乱。在这个研究项目中，我们将重点关注三个关键领域：1)。斑马鱼Ccms发病过程中gfp标记血管内皮细胞的分离2）. 通过对斑马鱼Ccm发病过程中血管内皮细胞的多基因组和蛋白质组学分析，阐明Ccm基因在血管生成/血管生成中的作用；3)鉴定微血管生成过程中与krit1、MGC4607、PDCD10相互作用的其他细胞因子。这项动物研究将提供关于血管生成过程中CCM基因及其复合物在血管内皮细胞中的细胞作用的详细知识，这是发展微血管畸形非手术治疗策略的关键的第一步。

DESCRIPTION (provided by applicant): This project aims to elucidate pathogenetic mechanisms in cerebral cavernous malformations (CCMs), a clinical syndrome in the brain and spinal cord marked by venous sinusoids that predispose to intracranial hemorrhage. Three genes have been identified as causes of CCMs; KRIT1 (CCM1), MGC4607 (CCM2), and PDCD10 (CCM3), which when mutated leads to the formation of neurovascular malformations. We propose that CCM genes regulate beta1 integrin-mediated signaling pathways during angiogenesis, remodeling, and/or homeostasis of the microvasculature and that perturbation of this process contributes to the development of CCMs. We have specifically silenced krit1 expression in cultured human vascular endothelial cells and observed dramatic perturbations in cellular proliferation, motility, and survival. We have also demonstrated: 1) interaction of krit1 with icap1alpha, a known mediator of beta1 integrin signaling; 2) interaction of krit1 with MGC4607; 3) perturbation of cultured endothelial cell proliferation, migration, and survival accompanied by reduced signaling through focal adhesion kinase (FAK), Rac1, and integrin-linked kinase (ILK) cascades, respectively, after siRNA-mediated silencing of either krit1 or icap1alpha; 4) enhancement of multiple beta1 integrin signaling cascades through targeting icap1 to the cell membrane; and 5) failed nuclear localization and degradation of icap1alpha and MGC4607 after silencing of krit1; 6) well documented cardiovascular defects of Ccm1 (santa, san) and Ccm2 (valentine, vtn) zebrafish, and 7) perturbation of vasculatures due to increased vascular endothelial cell apoptosis in san zebrafish. In this research project, we will focus on three critical areas: 1). Isolation of the GFP-labeled vascular endothelial cells during pathogenesis of zebrafish Ccms; 2). The elucidation of the role of Ccm genes in the angiogenesis/vasculogenesis with multiple genomic and proteomic analysis of the vascular endothelial cells during pathogenesis of zebrafish CCMs; and 3) identification of other cellular factors interacting of krit1, MGC4607, and PDCD10 during microvascular angiogenesis. This animal research will provide detailed knowledge regarding the cellular role of CCM genes and their complex in vascular endothelial cells during angiogenesis, a critical first step in the development of nonsurgical therapeutic strategies for microvascular malformations. PUBLIC HEALTH RELEVANCE: Cerebral cavernous malformations (CCMs) is a vascular syndrome in the brain that causes stroke. CCMs occur in approximately 0.5% of the population and represent 10% of all vascular malformations. Since CCM is the major cause for seizures among Hispanic ethnic group which contributes to over 85% population in El Paso, our proposed genomic and proteomic study on animal model is not only essential for understanding the molecular pathogenesis of this disease, but also extremely important for regional public health.