Department of Pediatrics, University of Colorado and Children's Hospital Colorado, Aurora, CO.
University of Colorado, Anschutz Medical Campus, Aurora, CO.
Blood. 2022 Apr 7;139(14):2240-2251. doi: 10.1182/blood.2021010683.
von Willebrand factor (VWF) plays a key role in normal hemostasis, and deficiencies of VWF lead to clinically significant bleeding. We sought to identify novel modifiers of VWF levels in endothelial colony-forming cells (ECFCs) using single-cell RNA sequencing (scRNA-seq). ECFCs were isolated from patients with low VWF levels (plasma VWF antigen levels between 30 and 50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were used as an additional control cell line. Cells were characterized for their Weibel Palade body (WPB) content and VWF release. scRNA-seq of all cell lines was performed to evaluate for gene expression heterogeneity and for candidate modifiers of VWF regulation. Candidate modifiers identified by scRNA-seq were further characterized with small-interfering RNA (siRNA) experiments to evaluate for effects on VWF. We observed that ECFCs derived from patients with low VWF demonstrated alterations in baseline WPB metrics and exhibit impaired VWF release. scRNA-seq analyses of these endothelial cells revealed overall decreased VWF transcription, mosaicism of VWF expression, and genes that are differentially expressed in low VWF ECFCs and control endothelial cells (control ECs). An siRNA screen of potential VWF modifiers provided further evidence of regulatory candidates, and 1 such candidate, FLI1, alters the transcriptional activity of VWF. In conclusion, ECFCs from individuals with low VWF demonstrate alterations in their baseline VWF packaging and release compared with control ECs. scRNA-seq revealed alterations in VWF transcription, and siRNA screening identified multiple candidate regulators of VWF.
血管性血友病因子 (VWF) 在正常止血中起着关键作用，VWF 的缺乏会导致临床上显著的出血。我们试图通过单细胞 RNA 测序 (scRNA-seq) 鉴定内皮祖细胞 (ECFCs) 中 VWF 水平的新型调节剂。ECFCs 从 VWF 水平低的患者（血浆 VWF 抗原水平在 30 至 50 IU/dL 之间）和健康对照中分离出来。人脐静脉内皮细胞被用作另一种对照细胞系。对细胞进行 Weibel-Palade 体 (WPB) 含量和 VWF 释放的特征分析。对所有细胞系进行 scRNA-seq，以评估基因表达异质性和 VWF 调节的候选调节剂。通过 scRNA-seq 鉴定的候选调节剂进一步通过小干扰 RNA (siRNA) 实验进行表征，以评估对 VWF 的影响。我们观察到，来自 VWF 水平低的患者的 ECFCs 表现出基线 WPB 指标的改变，并表现出 VWF 释放受损。对这些内皮细胞进行 scRNA-seq 分析显示，VWF 转录总体减少，VWF 表达的镶嵌性，以及在低 VWF ECFCs 和对照内皮细胞（对照 ECs）中差异表达的基因。潜在 VWF 调节剂的 siRNA 筛选提供了更多的调节候选者的证据，其中 1 个候选者 FLI1 改变了 VWF 的转录活性。总之，与对照 ECs 相比，来自 VWF 水平低的个体的 ECFCs 在其基线 VWF 包装和释放方面表现出改变。scRNA-seq 显示 VWF 转录发生改变，siRNA 筛选鉴定了多个 VWF 的候选调节因子。
