Molecular and cellular etiology of cerebral cavernous malformations

描述(申请人提供)：本项目旨在阐明脑海绵状畸形(CCM)的发病机制，CCM是一种脑和脊髓的临床综合征，以静脉窦为特征，易发生颅内出血。有三个基因被确定为CCMS的病因：KRIT1(CCM1)、MGC4607(CCM2)和PDCD10(CCM3)，当这些基因突变时，会导致神经血管畸形的形成。我们认为CCM基因在微血管系统的血管生成、重塑和/或动态平衡过程中调节β1整合素介导的信号通路，这一过程的扰动有助于CCM的发展。我们已经特异性地沉默了培养的人血管内皮细胞中Krit1的表达，并观察到细胞增殖、运动和存活的戏剧性扰动。我们还证实：1)krit1与已知的β1整合素信号中介体icap1α的相互作用；2)krit1与MGC4607的相互作用；3)在siRNA介导的krit1或icap1α沉默后，分别通过粘着斑激酶(FAK)、rac1和整合素连接激酶(Ilk)抑制培养的内皮细胞的增殖、迁移和存活并伴随信号的减少；4)通过靶向icap1到细胞膜来增强多个beta1整合素信号通路；以及5)krit1沉默后icap1α和MGC4607的核定位和降解失败；6)Ccm1(Santa，San)和CCM2(瓦伦丁，VTN)斑马鱼的心血管缺陷已有充分证据，以及7)San斑马鱼由于血管内皮细胞凋亡增加而导致的血管紊乱。在本研究项目中，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；以及3)鉴定Krit1、MGC4607和PDCD10在微血管形成过程中相互作用的其他细胞因子。这项动物研究将提供关于CCM基因及其复合体在血管生成过程中血管内皮细胞中的细胞作用的详细知识，这是开发微血管畸形非手术治疗策略的关键第一步。 与公共卫生相关：脑海绵状畸形(CCM)是一种大脑中的血管综合征，会导致中风。CCMS发生在大约0.5%的人群中，占所有血管畸形的10%。由于CCM是西班牙裔人群癫痫发作的主要原因，西班牙裔人群占埃尔帕索人口的85%以上，因此我们提出的动物模型基因组和蛋白质组研究不仅对于了解这种疾病的分子发病机制至关重要，而且对地区公共卫生也非常重要。

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.