The Role of Vascular Flt-1 in Endothelial-Pericyte Interactions

项目总结 血管形成需要一组对信号有不同反应的内皮细胞 投入。在发育过程中，内皮细胞对血管内皮生长因子的反应不同 (VEGF)-采用网络扩展所需表型的信令。血管发育异常 与肿瘤形成或糖尿病视网膜病变等病理情况相关的部分原因可能是 失去受调控的内皮异质性。血管内皮生长因子受体Flt-1(VEGFR-1)参与网络形成 通过可溶性异构体(sflt-1)的异质性表达，进而在空间上调节血管内皮生长因子信号转导 为新生的导管萌芽提供当地萌芽指导(Cappell等人，2009年)。表型异质性的研究 血管发育中的内皮细胞可能对血管发育的其他方面很重要，例如 内皮与血管周围细胞的相互作用称为周细胞。周细胞提供结构稳定性以 血管成熟和内皮细胞-周细胞相互作用的紊乱导致血管病变。 因此，推测内皮细胞表型异质性是由flt-1调节的。 血管内皮生长因子信号，以及这种异质性的各个方面促进了适当的内皮-周细胞相互作用。 因此，这项研究的一个主要目的是研究Flt-1如何在空间上调节内皮细胞 细胞异质性，以在体内建立适当的血管形态发生。将观察血管形态。 利用Flt-1条件缺失小鼠建立嵌合型Flt-1表达小鼠视网膜。体内和体内 然后，体外观察将被用来生成Flt-1在调节 血管内皮细胞表型的异质性和整体血管形态。此外，Flt-1在人类免疫系统中的作用 空间调控内皮细胞-周细胞的关联将通过体外试验进行探索。在胚胎干细胞中 (ES)细胞来源的血管、血管内皮生长因子信号将通过基因操作Flt-1的表达而受到干扰。 将评估内皮细胞-周细胞相互作用以表征周细胞募集的空间调节 和投资。改变Flt-1表达的空间分布对内皮细胞-周细胞的影响 相互作用，由野生型(WT)和Flt-1突变细胞组成的马赛克血管将被评估为周细胞 投资。模拟Flt-1促进血管内皮细胞异质性的计算模型 将建立调控周细胞-内皮细胞相互作用的工具，以了解 Flt-1表达中断的后果(例如，肿瘤环境)。来自体外实验的观察将会 在Silico模型中指导该模型的构建和测试。最后，Flt-1的调控机制 将描述体内周细胞-内皮细胞的相互作用。发育中的Flt-1的视网膜血管形成 将对条件缺失小鼠进行镶嵌Flt-1表达和周细胞投资的评估。模拟 由Flt-1对周细胞结合的调节的计算机模型产生的将提供一种手段 解释、分析和推进实验观察和方法。

PROJECT SUMMARY Blood vessel formation requires a group of endothelial cells with heterogeneous responses to signaling inputs. During development, endothelial cells respond differentially to vascular endothelial growth factor (VEGF)-A signaling to adopt phenotypes required for network expansion. Abnormal vascular development associated with pathological conditions such as tumorigenesis or diabetic retinopathy likely results in part from loss of regulated endothelial heterogeneity. VEGF receptor Flt-1 (VEGFR-1) contributes to network formation via heterogeneous expression of the soluble isoform (sFlt-1) that in turn spatially regulates VEGF signaling to provide local sprout guidance to emerging vessel sprouts (Chappell et al, 2009). Phenotypic heterogeneity of endothelial cells in developing vessels is likely important for other aspects of vascular development, such as endothelial interactions with perivascular cells known as pericytes. Pericytes provide structural stability to maturing vessels, and perturbations in endothelial-pericyte interactions contribute to vascular pathologies. Thus, it is intriguing to speculate that endothelial phenotypic heterogeneity is modulated by Flt-1 regulation of VEGF signaling, and that aspects of this heterogeneity facilitate proper endothelial-pericyte interactions. One primary objective of this study therefore is to investigate how Flt-1 spatially regulates endothelial cell heterogeneity to establish proper vascular morphogenesis in vivo. Vascular morphology will be observed in developing mouse retinas with mosaic flt-1 expression via use of flt-1 conditional deletion mice. In vivo and in vitro observations will then be used to generate a computational model for Flt-1 activity in regulating the phenotypic heterogeneity of endothelial cells and overall vessel morphology. In addition, the role of Flt-1 in spatially regulating endothelial-pericyte associations will be explored with in vitro assays. In embryonic stem (ES) cell-derived vessels, VEGF signaling will be perturbed via genetic manipulation of flt-1 expression. Endothelial-pericyte interactions will be evaluated to characterize the spatial regulation of pericyte recruitment and investment. To assess the effect of altered spatial distribution of flt-1 expression on endothelial-pericyte interactions, mosaic vessels composed of wild-type (WT) and flt-1 mutant cells will be evaluated for pericyte investment. A computational model simulating how Flt-1 promotes vessel endothelial cell heterogeneity to regulate pericyte-endothelial cell interactions will be created as a tool to understand the biological consequences of disruptions in flt-1 expression (e.g. tumor setting). Observations from in vitro experiments will guide the construction and testing of this in silico model. Lastly, the mechanisms by which Flt-1 regulates pericyte-endothelial interactions in vivo will be characterized. Retinal vasculature from developing flt-1 conditional deletion mice will be evaluated for mosaic flt-1 expression and investment of pericytes. Simulations generated by the computer model for Flt-1 regulation of pericyte associations will provide a means for interpreting, analyzing, and advancing experimental observations and approaches.