CRISPR/Cas9-mediated Gene Knockout Reveals a Guardian Role of NF-kB/RelA in Maintaining the Homeostasis of Human Vascular Cells

Dysfunction of blood vessels leads to severe vasculature pathogenesis. Previous studies have demonstrated that constitutive NFkB activation results in chronic vascular inflammation, leading to various cardiovascular diseases. However, how NFkB regulates blood vessel homeostasis remains largely elusive. Here, using CRISPR/Cas9-mediated gene editing, we generated RelA knockout human embryonic stem cells (hESCs) and differentiated them into human vascular derivatives to study how NFkB modulates vascular cells under basal and inflammatory conditions. Multi-dimensional phenotypic assessments and transcriptomic analyses revealed that RelA deficiency affected vascular cells via modulating vascular inflammation, survival, vasculogenesis, differentiation and extracellular matrix organization in a cell type-specific manner under basal condition, and that RelA protected vascular cells against apoptosis and modulated vascular inflammatory response upon TNFa stimuli. Lastly, further evaluation of gene expression patterns in IkBa knockout vascular cells demonstrated that IkBa acted largely independent of NFkB signaling pathway. Taken together, our data reveals a protective role of NFkB/RelA in modulating human blood vessel homeostasis and maps the human vascular transcriptomic landscapes for the discovery of novel therapeutic targets. Overall design: Transcriptomic analyses of WT, RelA and IkBa deficiency vascular cells under basal and inflammatory condition.血管功能异常可引发严重的血管病变。既往研究证实，组成型核因子κB（NFκB）激活会导致慢性血管炎症，进而引发多种心血管疾病。然而，NFκB如何调控血管稳态在很大程度上仍未明确。本研究采用CRISPR/Cas9介导的基因编辑技术，构建了RelA敲除的人胚胎干细胞（human embryonic stem cells, hESCs），并将其定向分化为人类血管衍生细胞，以探究NFκB在基础及炎症状态下对血管细胞的调控机制。多维度表型评估与转录组分析显示，在基础状态下，RelA缺失通过细胞类型特异性的方式调控血管炎症、细胞存活、血管生成、细胞分化及细胞外基质组织，进而影响血管细胞功能；在肿瘤坏死因子α（Tumor necrosis factor α, TNFα）刺激下，RelA可保护血管细胞免受凋亡损伤，并调控血管炎症反应。此外，对IkBa敲除血管细胞的基因表达模式进行进一步评估后发现，IkBa的功能很大程度上不依赖于NFκB信号通路。综上，本研究揭示了NFκB/RelA在调控人类血管稳态中的保护作用，并绘制了人类血管转录组图谱，为新型治疗靶点的发现提供了重要依据。整体实验设计：在基础及炎症状态下，对野生型（wild type, WT）、RelA敲除及IkBa敲除的血管细胞开展转录组分析。