Novel growth factor and signaling requirements for human capillary tube assembly

 描述（由申请人提供）：在这项新提案中，我们调查了我们的发现，定义了控制EC小管发生和EC-周细胞管共组装的新型生长因子组合和信号通路，这是创建毛细血管网络所必需的，这对组织灌注至关重要，发育和功能维持。毛细血管由两种主要的细胞类型组成，EC和周细胞，它们共同组装以形成极化的EC内衬管，其中周细胞位于腔外，基底膜基质介于其间。Davis实验室率先开发了使用人类EC和周细胞的体外系统，该系统在无血清定义条件下在3D基质中模拟这些事件。值得注意的是，我们已经确定，添加五种确定的生长因子，SCF，IL-3，SDF-1 α，FGF-2和胰岛素（因子），允许人EC小管形成和周细胞的募集伴随血管基底膜基质组装，这是我们实验室首次报道的发现。新的初步数据表明，这些因子及其受体通过激活三个关键的协同信号通路来控制小管发生; PI 3 K/Akt/mTor，Raf/Mek/Erk和Jak/Stat。这些途径被因子以其独特的方式强烈激活并随时间持续，并且是EC小管形成所必需的;并且这种重要的信号传导在VEGF、FGF-2或VEGF+FGF-2添加后不明显。此外，我们定义的系统导致了另一个主要的见解，证明了VEGF作为因子诱导的EC微管发生和EC-周细胞管共组装的上游引物的新作用，而它不能直接刺激这些过程。我们还报道了EC衍生的PDGF-BB和HB-EGF在介导周细胞募集、增殖和基底膜形成中起关键作用，这一观察结果在体内使用发育中的鹌鹑胚胎得到证实。最后，我们获得了新的证据，证明EC衍生的TGF β 1、激活素B、CTGF和CCL 2家族趋化因子在EC-周细胞管共组装中的作用。总之，这些新的见解提供了一个分子路线图，解剖如何定义的生长因子，受体，其共同的适配器，和重叠的信号通路控制EC tubulogenesis和EC-周细胞管共组装使用在体外和体内的实验方法。我们提出了三个具体的目标，以进一步研究这些新的见解的基本过程中的毛细管组装在体外和体内响应于定义的生长因子，他们是：目的1：确定和表征所需的SCF，IL-3，SDF-1a和FGF-2-和受体依赖的信号转导事件，控制EC tubulogenesis在体外和体内。目标2：阐明因子受体和衔接子如何协同作用以激活信号通路Akt/mTor、Raf/Erk和Jak/Stat，从而导致EC小管形成的潜在机制。目标3：阐明SCF、IL-3、SDF-1a和FGF-2以及受体依赖性信号传导如何导致EC-周细胞管共组装和血管基底膜形成。

 DESCRIPTION (provided by applicant): In this new proposal, we investigate our findings defining novel growth factor combinations and signaling pathways that control EC tubulogenesis and EC-pericyte tube co-assembly, which are necessary to create capillary networks, which critically support tissue perfusion, development and functional maintenance. Capillaries consist of two major cell types, ECs and pericytes, which co-assemble to form polarized EC-lined tubes with abluminally positioned pericytes and an intervening basement membrane matrix. The Davis lab has pioneered the development of in vitro systems using human ECs and pericytes, which model these events in 3D matrices under serum-free defined conditions. Remarkably, we have identified that addition of five defined growth factors, SCF, IL-3, SDF-1a, FGF-2, and insulin (Factors), allows for human EC tubulogenesis and recruitment of pericytes with accompanying vascular basement membrane matrix assembly, a finding first reported by our laboratory. Novel preliminary data reveals that these Factors and their receptors control tubulogenesis through activation of three key synergistic signaling pathways; PI3K/Akt/mTor, Raf/Mek/Erk, and Jak/Stat. These pathways are strongly activated and sustained over time by the Factors in a manner that is unique to them, and which are necessary for EC tubulogenesis; and this important signaling is not manifest following VEGF, FGF-2 or VEGF+FGF-2 addition. Furthermore, our defined system led to another major insight demonstrating a novel role for VEGF as an upstream primer of Factor-induced EC tubulogenesis and EC-pericyte tube co-assembly, while it fails to stimulate these processes directly. We also reported that EC- derived PDGF-BB and HB-EGF play a key role in mediating pericyte recruitment, proliferation and basement membrane formation, an observation that was confirmed in vivo using developing quail embryos. Finally, we have obtained novel evidence for a role for EC-derived TGFß1, activinB, CTGF, and CCL2 family chemokines in EC-pericyte tube co-assembly. Together, these new insights provide a molecular road map to dissect how defined growth factors, receptors, their common adapters, and overlapping signaling pathways control EC tubulogenesis and EC-pericyte tube co-assembly using both in vitro and in vivo experimental approaches. We propose three specific aims to further investigate these novel insights into the fundamental process of capillary tube assembly in vitro and in vivo in response to defined growth factors and they are; Aim1: To identify and characterize required SCF, IL-3, SDF-1a and FGF-2- and receptor-dependent signaling events that control EC tubulogenesis in vitro and in vivo. Aim2: To elucidate the underlying mechanisms how Factor receptors and adapters synergistically work together to activate the signaling pathways, Akt/mTor, Raf/Erk, and Jak/Stat, leading to EC tubulogenesis. Aim3: To elucidate how SCF, IL-3, SDF-1a and FGF-2- and receptor-dependent signaling leads to EC- pericyte tube co-assembly and vascular basement membrane formation.