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

描述(申请人提供)：血管形成需要一组对信号输入具有不同反应的内皮细胞。在发育过程中，内皮细胞对血管内皮生长因子-A信号有不同的反应，以采用网络扩展所需的表型。与肿瘤形成或糖尿病视网膜病变等病理条件相关的血管异常发育可能部分是由于受调控的内皮异质性丧失所致。血管内皮生长因子受体Flt-1(VEGFR-1)通过可溶性异构体(sFlt-1)的异质性表达促进网络的形成，而sFlt-1反过来在空间上调节VEGF信号，为新生的血管萌芽提供局部指导(Cappell等人，2009)。血管发育中内皮细胞的表型异质性可能对血管发育的其他方面很重要，例如内皮细胞与血管周围细胞的相互作用。周细胞为成熟的血管提供结构稳定性，内皮细胞-周细胞相互作用的扰动有助于 血管病理学。因此，推测内皮细胞表型的异质性是很有趣的。 受Flt-1对血管内皮生长因子信号的调节，这种异质性的某些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标 目的是研究Flt-1如何在空间上调节内皮细胞的异质性，从而在体内建立适当的血管形态发生。通过使用Flt-1条件缺失小鼠，将在发育中观察到嵌合型Flt-1表达的小鼠视网膜的血管形态。然后，体内和体外观察将被用来生成Flt-1在调节内皮细胞表型异质性和整体血管形态方面的活性的计算模型。此外，Flt-1在空间调控内皮细胞-周细胞关联中的作用将在病毒检测中进行探索。在胚胎干细胞来源的血管中，通过基因操作Flt-1的表达，血管内皮生长因子信号会受到干扰。将评估内皮细胞-周细胞相互作用，以表征周细胞招募和投资的空间调节。为了评估Flt-1表达的空间分布改变对内皮细胞-周细胞相互作用的影响，将评估由野生型(WT)和Flt-1突变细胞组成的镶嵌血管的周细胞投资。将建立一个模拟Flt-1如何促进血管内皮细胞异质性以调节周细胞-内皮细胞相互作用的计算模型，作为了解Flt-1表达中断(例如，肿瘤发生)的生物学后果的工具。来自体外实验的观察将指导这一硅胶模型的构建和测试。最后，Flt-1在体内调节周细胞-内皮细胞相互作用的机制将被描述。发育中的Flt-1条件缺失小鼠的视网膜血管系统将被评估Flt-1的嵌合体表达和周细胞的投资。由Flt-1调控周细胞结合的计算机模型产生的模拟将为解释、分析和推进实验观察和方法提供一种手段。

DESCRIPTION (provided by applicant): 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 vito 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 viro 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.