Single-Cell Receptor Quantification of an In Vitro Coculture Angiogenesis Model Reveals VEGFR, NRP1, Tie2, and PDGFR Regulation and Endothelial Heterogeneity
Single-Cell Receptor Quantification of an In Vitro Coculture Angiogenesis Model Reveals VEGFR, NRP1, Tie2, and PDGFR Regulation and Endothelial Heterogeneity
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DOI：
10.3390/pr7060356
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发表时间：
2019-06
期刊：
影响因子：
3.5
通讯作者：
Si Chen;P. Imoukhuede
中科院分区：
文献类型：
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作者：
Si Chen;P. Imoukhuede
血管生成，即在原有血管的基础上形成新的血管，对正常发育和许多病理过程都是必不可少的。系统生物学通过分析调节血管生成过程的酪氨酸激酶受体(RTK)并通过计算模拟RTK信号通路，为研究血管生成提供了一种独特的方法。历史上，这种系统生物学方法被应用于体外血管生成检测，然而，这些检测方法很难量化，并且其时间分析的潜力有限。在这项研究中，我们采用了一种简单的由人脐静脉内皮细胞(HUVECs)和人真皮成纤维细胞(HDFS)组成的二维血管生成实验，并检测了一组六个RTK的时间动力学和长达17天的细胞异质性。在单独培养不表达VEGFR的培养24小时的HDF质膜上，我们观察到约2700个VEGFR1(血管内皮生长因子受体1)。我们观察到，在内皮细胞形成过程中，共培养的人脐静脉内皮细胞质膜上每个细胞有4000-8100个VEGFR2。我们发现共培养的人脐静脉内皮细胞膜上的血小板衍生生长因子受体(PDGFRs)稳步增加，更有趣的是，在共培养的前6小时内，人脐静脉内皮细胞膜上有1,900-2,900个PDGFRβ。这些定量的发现将为我们深入了解血管生成过程中的分子调控，并有助于评估体外管形成模型及其生理相关性。
Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for both normal development and numerous pathologies. Systems biology has offered a unique approach to study angiogenesis by profiling tyrosine kinase receptors (RTKs) that regulate angiogenic processes and computationally modeling RTK signaling pathways. Historically, this systems biology approach has been applied on ex vivo angiogenesis assays, however, these assays are difficult to quantify and limited in their potential of temporal analysis. In this study, we adopted a simple two-dimensional angiogenesis assay comprised of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) and examined temporal dynamics of a panel of six RTKs and cell heterogeneity up to 17 days. We observed ~2700 VEGFR1 (vascular endothelial growth factor receptor 1) per cell on 24-h-old cocultured HDF plasma membranes, which do not express VEGFR when cultured alone. We observed 4000–8100 VEGFR2 per cell on cocultured HUVEC plasma membranes throughout endothelial tube formation. We showed steady increase of platelet-derived growth factor receptors (PDGFRs) on cocultured HDF plasma membranes, and more interestingly, 1900–2900 PDGFRβ per plasma membrane were found on HUVECs within the first six hours of coculturing. These quantitative findings will offer us insights into molecular regulation during angiogenesis and help assess in vitro tube formation models and their physiological relevance.