Transcriptional profiling of ex vivo isolated mouse blood vascular and lymphatic endothelial cells

The lymphatic vascular system plays important roles in the maintenance of interstitial fluid pressure, the afferent immune response and the absorption of dietary lipids. However, the molecular mechanisms that control lymphatic vessel network maturation and function remain largely unknown. To identify novel players in lymphatic vessel function, we isolated pure populations of lymphatic and blood vascular endothelial cells from mouse intestine using fluorescence-activated high-speed cell sorting and performed transcriptional profiling. We found that the axonal guidance molecules semaphorin 3A (Sema3A) and Sema3D were specifically expressed by lymphatic vessels. Quantitative PCR of ex vivo isolated cells and immunohistochemical analysis confirmed these results. Importantly, we found that the semaphorin receptor neuropilin-1 (Nrp-1) is expressed on the valves of collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1, but not with an antibody that blocks VEGFA binding to Nrp-1, resulted in abnormal development of collecting lymphatic vessels and valves, and aberrant smooth muscle cell coverage. Conversely, Sema3A-deficient mice displayed branching defects of collecting lymphatic vessels as well as impaired valve development. Together, these results reveal an unanticipated role of Sema3A/Nrp-1 signaling in the maturation of the lymphatic vascular network. Colon single-cell suspensions were prepared by a fast protocol that minimizes the RNA degradation. Fluorescence-activated cell sorting (FACS) was used to sort blood vascular endothelial cells (BEC) and lymphatic endothelial cells (LEC). 4 animal-matched pairs of LEC and BEC were chosen based on the quality of extracted and amplified material to provide homogenous groups of biological replicates. This gave 8 samples to analyze. Samples present LEC and BEC isolated from 4 healthy normal mice. The 4 mice used present the 4 biological replicates.淋巴血管系统在维持组织液压力、介导传入免疫应答以及膳食脂质吸收过程中发挥关键作用。然而，调控淋巴管网络成熟与功能的分子机制在很大程度上仍未明确。为鉴定淋巴管功能的新型调控因子，我们通过荧光激活高速细胞分选（fluorescence-activated high-speed cell sorting）从小鼠肠道中分离得到纯度均一的淋巴管内皮细胞与血管内皮细胞，并开展转录组分析。我们发现轴突导向分子信号素3A（semaphorin 3A, Sema3A）与信号素3D（Sema3D）可特异性表达于淋巴管。针对离体分离细胞的定量PCR检测与免疫组织化学分析验证了这一结果。尤为重要的是，我们发现信号素受体神经纤毛蛋白-1（neuropilin-1, Nrp-1）在集合淋巴管的瓣膜表面表达。在妊娠小鼠的子宫内（胚胎发育阶段E12.5至E16.5）施用可阻断Sema3A与Nrp-1结合的抗体，而非施用阻断血管内皮生长因子A（VEGFA）与Nrp-1结合的抗体，可引发集合淋巴管与瓣膜发育异常，同时出现平滑肌细胞覆盖异常的表型。反之，Sema3A缺陷小鼠则表现出集合淋巴管分支缺陷以及瓣膜发育受损的表型。综上，本研究结果揭示了Sema3A/Nrp-1信号通路在淋巴管网络成熟过程中此前未被发现的重要作用。本研究采用可最小化RNA降解的快速方案制备结肠单细胞悬液，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分离得到血管内皮细胞（blood vascular endothelial cells, BEC）与淋巴管内皮细胞（lymphatic endothelial cells, LEC）。基于提取与扩增产物的质量，我们选取4对动物匹配的LEC与BEC样本，以构建均质的生物学重复组，最终获得8份待分析样本。所有样本均来自4只健康正常小鼠的LEC与BEC，这4只小鼠即作为4个生物学重复。