BACKGROUND Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. METHODS Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. RESULTS Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CONCLUSIONS CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.

中文翻译：

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脑内皮祖细胞中的 mTORC1 信号传导有助于 CCM 发病机制。


背景脑血管畸形（CCM）主要发现于大脑内，导致中风、癫痫发作和局灶性神经功能缺损的风险增加。脑血管系统的独特特征是由脑神经血管单元形成的血脑屏障。最近的研究表明，CCM 基因的丢失会导致血脑屏障完整性破坏，从而成为 CCM 发展的刺激事件。 CCM 病变最初被认为源自血管生成静脉毛细血管内皮细胞 (EC) 和各自的内皮祖细胞 (EPC) 子集的单克隆扩增。然而，脑内皮细胞/内皮祖细胞亚类中对于 CCM 病变发生和进展的关键信号传导事件尚不清楚。方法通过将 Pdcd10fl/fl 小鼠与 Mfsd2a-CreERT2 小鼠杂交产生脑 EC 特异性 CCM3 缺陷 (Pdcd10BECKO) 小鼠。单细胞 RNA 测序分析由 chromium 单细胞平台（10× 基因组学）进行。根据目视检查和 GO 分析，将细胞簇注释为 EC 亚型。通过 2 光子体内成像和组织免疫荧光分析使脑血管可视化。通过细胞生物学和生化方法，通过 CCM3 和 Cav1 (caveolin-1) 对 mTOR（雷帕霉素的机械靶标）信号进行调节。结果对具有可见 CCM 病变的 P10 Pdcd10BECKO 小鼠进行单细胞 RNA 测序分析，发现 CCM 病变特征和有丝分裂 EC 簇上调，但血脑屏障相关 EC 簇减少。然而，在 P6 Pdcd10BECKO 大脑的早期阶段就发现了一个独特的 EPC 簇，该簇具有高表达水平的干细胞标记物，富含 mTOR 信号传导。 事实上，mTOR 信号传导在小鼠和人类 CCM 病变中均上调。 Raptor（mTOR 的调节相关蛋白）的遗传缺陷，而不是 Rictor（mTOR 的雷帕霉素不敏感伴侣）的遗传缺陷，阻止了 Pdcd10BECKO 模型中 CCM 病变的形成。重要的是，mTORC1（mTOR 复合物 1）药理学抑制剂雷帕霉素抑制 Pdcd10BECKO 小鼠中的 EPC 增殖并改善 CCM 发病机制。机制研究表明，CCM3 耗尽的 EPC 介导的细胞内运输和 mTORC1 信号蛋白复合物形成中 Cav1/caveolae 增加。结论 CCM3 对于维持血脑屏障完整性至关重要，并且 CCM3 缺失诱导的大脑 EPC 中的 mTORC1 信号传导启动并促进 CCM 发病机制。