A spatial vascular transcriptomic, proteomic and phosphoproteomic atlas unveils an angiocrine Tie Wnt signaling axis in the liver

Single cell transcriptomic analyses (scRNAseq) of hepatocytes and liver endothelial cells (L-EC) have revolutionized the understanding of the spatial architecture of liver structure and function. The spatial alignment of L-EC and hepatocytes is pivotal for liver function in health and disease given that L-EC act as instructive gatekeeper of nearby hepatocytes including the maintenance of liver metabolic zonation in a Wnt-dependent manner. Advancing liver biology beyond the ’transcript-centric’ view of scRNAseq analyses is presently restricted by the limited resolution of proteomics and genome-wide techniques to analyse post-translational modifications. Here, by combining spatial cell sorting methodology with transcriptomic and quantitative proteomic/phospho-proteomic analyses, we established the first functionally and spatially-resolved proteome landscape of the liver endothelium, yielding deep mechanistic insight into zonated vascular signalling mechanisms. Phosphorylation of receptor tyrosine kinases (RTK) was detected preferentially in the central vein area resulting in an atypical enrichment of tyrosine phosphorylation. Prototypic biological validation of the identified strong phosphorylation gradient of the vascular RTK Tie1 by blockade resulted in the rapid peri-central dysregulation of the L-EC transcriptome. Notably, the expression of Wnt9b in L-EC was discovered as Tie receptor controlled with reciprocal regulation by FoxO1 and STAT3 transcription factors. Genetic inactivation of Tie1 in L-EC or antibody blockade resulted in reduced liver regeneration following partial hepatectomy with reduced Wnt ligand and Wnt target gene expression, including Sox9, Tbx3 and Lgr5. Taken together, the study has yielded unparalleled insight into the spatial organization of L EC signalling and discovered a vascular Tie/Wnt signalling axis as regulator of liver function. The employed spatial sorting technique followed by phospho-proteomic analysis may be employed as a universally adaptable strategy for the spatial phosphoproteomic analysis of scRNAseq data-defined relevant cellular (sub)-populations.

肝细胞与肝内皮细胞（liver endothelial cells, L-EC）的单细胞转录组测序（single cell RNA sequencing, scRNAseq）分析，彻底革新了学界对肝脏结构与功能的空间组织模式的认知。鉴于肝内皮细胞可作为邻近肝细胞的调控性守门细胞，包括以Wnt依赖方式维持肝脏代谢分区，二者的空间定位对于健康与疾病状态下的肝脏功能均至关重要。当前，将肝脏生物学研究突破单细胞转录组测序的“转录组中心”视角，受限于蛋白质组学与全基因组技术在分析翻译后修饰时的分辨率不足。本研究将空间细胞分选方法与转录组学、定量蛋白质组/磷酸化蛋白质组分析相结合，构建了首个兼具功能与空间分辨率的肝脏内皮细胞蛋白质组图谱，为解析分区血管信号通路机制提供了深入的机理性见解。研究发现受体酪氨酸激酶（receptor tyrosine kinases, RTK）的磷酸化优先富集于中央静脉区域，进而导致酪氨酸磷酸化出现非典型富集。针对所鉴定出的血管RTK Tie1的强磷酸化梯度开展典型生物学验证，通过阻断该靶点可快速引发肝内皮细胞转录组的中央静脉周围失调。值得注意的是，本研究发现肝内皮细胞中Wnt9b的表达受Tie受体调控，并由FoxO1与STAT3转录因子进行双向调控。在肝内皮细胞中敲除Tie1或通过抗体阻断该靶点，可导致部分肝切除术后肝脏再生能力下降，同时伴随Wnt配体及Sox9、Tbx3、Lgr5等Wnt靶基因的表达水平降低。综上，本研究为肝脏内皮细胞信号通路的空间组织模式提供了前所未有的认知，并发现了血管Tie/Wnt信号轴作为肝脏功能的调控因子。本研究采用的空间分选结合磷酸化蛋白质组分析的策略，可作为通用适配方法，用于对单细胞转录组测序定义的相关细胞（亚）群开展空间磷酸化蛋白质组学分析。