Cerebral Cavernous Malformations Develop Through Clonal Expansion of Mutant Endothelial Cells.
Cerebral Cavernous Malformations Develop Through Clonal Expansion of Mutant Endothelial Cells.
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DOI：
10.1161/circresaha.118.313970
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发表时间：
2018-10-26
影响因子：
20.1
通讯作者：
Marchuk DA
中科院分区：
文献类型：
--
作者：
Detter MR;Snellings DA;Marchuk DA
血管畸形发生在全身的血管中。已经确定了许多这些疾病的致病基因突变；然而，对这些畸形中的突变细胞谱系知之甚少。我们利用脑海绵状畸形（CCMs）的诱导小鼠模型，结合多色荧光报告器来可视化突变内皮细胞（ECs）对畸形的贡献。我们将Ccm3小鼠模型与五彩纸屑荧光报告基因结合，同时删除Ccm3，并用四种可能的颜色之一标记突变EC。我们从一系列脑切片中获得z系列共聚焦图像，并创建了整个CCM的3D重建，以可视化CCM发育过程中的突变ECs。我们观察到一个明显的模式，CCMs与突变ECs排列，标记为单一五彩纸屑色（n=42）。通过最近邻分析确定的CCM内克隆优势ECs的密切3D分布与非CCM对照脑切片中ECs的背景彩纸屑标记以及计算机模拟具有统计学差异（p<0.001）。许多小的（<100μm直径）CCMs几乎完全由克隆优势突变型ECs组成，标记为相同的彩色，而大的（100μm直径）CCMs既包含克隆优势突变型细胞，也包含野生型ECs。我们提出了一种CCM发育模型，其中EC获得第二个体细胞突变，经历克隆扩增以启动CCM的形成，然后结合邻近的野生型EC以增加畸形的大小。这是第一个用单细胞分辨率观察突变ECs在CCMs内克隆扩增的研究。野生型ECs与生长畸形的结合呈现了另一系列细胞事件，其阐明将增强我们对CCMs的理解，并可能提供新的治疗机会。
Vascular malformations arise in vessels throughout the entire body. Causative genetic mutations have been identified for many of these diseases; however, little is known about the mutant cell lineage within these malformations. We utilize an inducible mouse model of cerebral cavernous malformations (CCMs) coupled with a multi-color fluorescent reporter to visualize the contribution of mutant endothelial cells (ECs) to the malformation. We combined a Ccm3 mouse model with the confetti fluorescent reporter to simultaneously delete Ccm3 and label the mutant EC with one of four possible colors. We acquired Z-series confocal images from serial brain sections and created 3D reconstructions of entire CCMs to visualize mutant ECs during CCM development. We observed a pronounced pattern of CCMs lined with mutant ECs labeled with a single confetti color (n=42). The close 3D distribution, as determined by the nearest neighbor analysis, of the clonally dominant ECs within the CCM was statistically different than the background confetti labeling of ECs in non-CCM control brain slices as well as a computer simulation (p<0.001). Many of the small (<100μm diameter) CCMs consisted, almost exclusively, of the clonally dominant mutant ECs labeled with the same confetti color whereas the large (>100μm diameter) CCMs contained both the clonally dominant mutant cells and wild type ECs. We propose of model of CCM development in which an EC acquires a second somatic mutation, undergoes clonal expansion to initiate CCM formation, and then incorporates neighboring wild type ECs to increase the size of the malformation. This is the first study to visualize, with single-cell resolution, the clonal expansion of mutant ECs within CCMs. The incorporation of wild type ECs into the growing malformation presents another series of cellular events whose elucidation would enhance our understanding of CCMs and may provide novel therapeutic opportunities.