Novel growth factor and signaling requirements for human capillary tube assembly

 描述(由申请人提供)：在这项新的提案中，我们调查了我们的发现，定义了控制EC小管形成和EC-周细胞管共同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，而毛细血管网络对组织的灌流、发育和功能维持至关重要。毛细血管由两种主要的细胞类型组成，内皮细胞和周细胞，它们共同组装形成极化的EC衬里的管，具有光亮的定位的周细胞和中间的基底膜基质。戴维斯实验室率先开发了使用人类内皮细胞和周细胞的体外系统，在无血清限定的条件下，以3D矩阵对这些事件进行建模。值得注意的是，我们已经确定了五种明确的生长因子，SCF、IL-3、SDF-1a、FGF-2和胰岛素(因子)的加入，允许人EC小管形成和周细胞招募伴随着血管基底膜基质组装，这是我们实验室首次报道的发现。新的初步数据显示，这些因子及其受体通过激活三个关键的协同信号通路：PI3K/Akt/mTor、Raf/MEK/Erk和Jak/Stat来控制小管形成。随着时间的推移，这些通路被这些因子以一种独特的方式强烈激活和维持，这些因子是EC小管形成所必需的；而这种重要的信号转导在加入VEGF、FGF2或VEGF+FGF2后并不明显。此外，我们定义的系统带来了另一个重要的洞察力，证明了VEGF作为因子诱导的EC小管形成和EC-周细胞管共同组装的上游启动子的新角色，但它无法直接刺激这些过程。我们还报道了EC来源的PDGF-BB和HB-EGF在介导周细胞招募、增殖和基底膜形成方面发挥关键作用，这一观察结果在体内发育的鹌鹑胚胎中得到了证实。最后，我们获得了新的证据，证明了EC来源的转化生长因子1、激活素B、CTGF和CCL2家族趋化因子在EC-周细胞管共同组装中所起的作用。总之，这些新的见解提供了一个分子路线图，用体外和体内的实验方法来剖析已定义的生长因子、受体、它们的共同适配器和重叠的信号通路如何控制EC小管发生和EC-周细胞管的共同组装。我们提出了三个具体的目标，以进一步研究这些新的洞察力在体外和体内毛细管组装的基本过程中对已定义的生长因子的响应，它们是：目的1：鉴定和表征所需的SCF、IL-3、SDF-1a和成纤维细胞生长因子-2及其受体依赖的信号事件，这些信号事件在体外和体内控制EC的小管形成。目的：阐明因子受体和适配器协同作用激活Akt/mTOR、Raf/Erk和Jak/Stat信号通路，导致EC小管形成的潜在机制。目的：阐明SCF、IL-3、SDF-1a、成纤维细胞生长因子-2及其受体依赖的信号转导途径在内皮细胞-周细胞管共装配和血管基底膜形成中的作用。

 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.