Endoglin Regulates Endothelial Survival and Capillary Tube Stability

描述(由申请人提供)：内皮细胞中的特定目标转化生长因子(TGF-2)超家族信号调节血管生成和血管形态形成的基本成分，包括增殖和毛细血管形成。转化生长因子-2超家族配体通过内皮细胞特异性的转化生长因子-2受体复合体ALK1(I型受体)和endoglin(共受体)以及普遍存在的I型转化生长因子-2受体ALK5分别激活典型的Smad1/5/8和Smad2/3通路而发挥调节作用。转化生长因子-2配体也通过非Smad信号通路，如MAPK和PI3K/Akt，尽管潜在的机制仍不清楚。Endoglin和ALK1在内皮细胞的转化生长因子-2信号转导中起着关键作用，它们的突变导致人类血管疾病，遗传性出血性毛细血管扩张(HHT1和2)，当Endoglin或ALK1被靶向缺失时，由于血管生成缺陷而导致的胚胎死亡表型，以及Endoglin在炎症和肿瘤诱导的血管生成过程中的高表达，都支持了Endoglin的关键作用。虽然endoglin的重要生物学作用已被确定，但endoglin在血管生物学中作用的分子基础仍不清楚。在这里，体外血管生成试验被用来精确地研究endoglin何时以及如何调节内皮细胞毛细血管的萌发和管子的形成。将endoglin缺失的内皮细胞与野生型内皮细胞进行比较发现，endoglin对其生理上相关的高亲和力配体-转化生长因子-2和骨形态发生蛋白-9的影响，对毛细血管萌发和毛细血管的稳定性有不同的调节作用。具体地说，转化生长因子-2主要通过抑制依赖内源性激素的Akt信号，导致毛细血管发芽和管状结构的退化。相反，endoglin增强Akt信号以响应BMP-9以促进毛细血管的稳定性。这些结果归因于endoglin和支架/运输蛋白GIPC之间的关联，因为破坏它们的相互作用消除了这种endoglin依赖的效应。鉴于我最近报道了通过GIPC和endoglin增强Smad 1/5/8信号，Akt和依赖endoglin的Smad 1/5/8信号之间存在潜在的串扰，我建议对此进行研究。最后，我发现了内源性endoglin和Akt之间的一种新的相互作用，这一发现可能会产生内源性endoglin生物学的新方面。基于这些初步数据，我提出以下假设：endoglin与GIPC相关，通过依赖BMP-9激活Akt来稳定内皮毛细血管，而通过依赖于TGF-2抑制Akt信号和细胞生存机制来破坏毛细血管的稳定，从而促进血管生成。这一假设将通过两个具体目标中概述的目标来解决。 与公共健康相关：我的应用程序的一个明显的创新方面是定义了endoglin在改变内皮细胞行为方面的新信号角色。目前的范式将endoglin描述为一种辅助的转化生长因子-2共受体，它调节两个相反的信号通路Smad1/5/8和Smad2/3的平衡，这两个信号通路分别引发促血管生成反应或抗血管生成反应[12-22]。然而，有几条证据挑战了endoglin的这种过于简单化的角色。首先，在携带纯合缺失的小鼠中观察到胚胎致死表型[1，2]。其次，在血管生成过程中，endoglin信号对Smad通路的意义还没有被完全理解，因为这些典型的信号转导可以在没有endoglin表达的情况下被激活[25]。我的初步发现表明，endoglin参与了其他重要的信号和细胞功能，如调节Akt激活和下游细胞生存机制。到目前为止，我已经确定了endoglin在其直接配体转化生长因子-2和骨形态发生蛋白-9的反应中，差异调节Akt激活的能力。因此，我的研究提案试图解决的一个非常有趣的方面是，endoglin如何识别两个结构相关的配体来引起对Akt激活的这种不同的影响。我的研究提案的另一个创新方面涉及计划使用体外模型系统，该系统包含许多(如果不是全部)血管生成过程的单独组件，如内皮细胞增殖、基底膜降解、迁移、排列和毛细管形成。几个小组已经使用细胞增殖和迁移分析来剖析endoglin调节血管生成的机制。然而，合适的体外系统，包括血管生成的大部分顺序阶段，将有利于监测血管生成进展过程中endoglin的作用。操纵各种条件，如配体治疗或改变蛋白质水平的影响，都是可能的。我已经开始使用这样的方法来研究endoglin函数。我基于毛细管形成实验的初步发现表明，endoglin在调节萌发的毛细管的稳定性方面发挥着重要作用，这一动态过程在很大程度上是由Akt信号和细胞生存机制控制的。在血管生成过程中，endoglin如何有助于这些成熟血管的稳定性是一个完全未被探索的研究领域，也是我研究计划的基础。通过使用几种新的体外血管生成分析方法，包括微载体和共培养方法，所提出的工作将对endoglin在血管生成过程中何时以及如何发挥其作用产生重要的洞察。

DESCRIPTION (provided by applicant): Specific Aims Transforming growth factor (TGF-2) super family signaling in endothelial cells regulates essential components of angiogenesis and vascular morphogenesis, including proliferation and capillary tube formation. TGF-2 super family ligands exert their regulatory effects through the endothelial cell specific TGF-2 receptor complex, ALK1 (type I receptor) and endoglin (co-receptor), along with the ubiquitous type I TGF-2 receptor, ALK5, to activate the canonical Smad 1/5/8 and Smad 2/3 pathways, respectively. TGF-2 ligands also signal through non-Smad pathways such as MAPKs and PI3K/Akt, although the underlying mechanisms remain obscure. A critical role for endoglin and ALK1 in TGF-2 signaling in endothelial cells is supported by their mutation resulting in the human vascular disease, hereditary hemorrhagic telangiectasia (HHT1 and 2), embryonic lethal phenotype due to defects in angiogenesis when either endoglin or ALK1 is targeted for deletion in mice, and by the elevated expression of endoglin during inflammation and tumor-induced angiogenesis. While important biological roles for endoglin have been established, the molecular basis for endoglin function in vascular biology remains poorly characterized. Here in vitro angiogenesis assays were employed to investigate precisely when and how endoglin regulates endothelial capillary sprouting and tube formation. Comparison of endoglin-null and wild type endothelial cells revealed that endoglin differentially regulates the stability of capillary sprouts and tubes in response to its physiologically relevant high-affinity ligands, TGF-2 and BMP-9. Specifically, TGF-2 resulted in regression of the capillary sprouts and tube structures, primarily through suppression of endoglin-dependent Akt signaling. Conversely, endoglin enhanced Akt signaling in response to BMP-9 to promote capillary stability. These outcomes are attributed to the association between endoglin and the scaffolding/trafficking protein, GIPC, since disrupting their interaction abrogated such endoglin-dependent effects. Given that I recently reported the enhancement of Smad 1/5/8 signaling through GIPC and endoglin, there exists a potential crosstalk between Akt and endoglin-dependent Smad 1/5/8 signaling, which I propose to investigate. Lastly, I discovered a novel interaction between endogenous endoglin and Akt, a finding that will likely yield new facets of endoglin biology. Based upon these preliminary data, I propose the following hypothesis: Endoglin associates with GIPC to promote angiogenesis by stabilizing endothelial capillaries via BMP-9-dependent Akt activation while destabilizing capillaries via TGF-2-dependent suppression of Akt signaling and cell survival mechanisms. This hypothesis will be addressed by the objectives outlined in two specific aims. PUBLIC HEALTH RELEVANCE: A distinctly innovative aspect of my application is in defining a new signaling role for endoglin in altering endothelial cell behavior. The current paradigm describes endoglin as an auxiliary TGF-2 co-receptor that regulates the balance of two opposing signaling pathways, Smad1/5/8 and Smad2/3, which invoke pro- or antiangiogenic responses, respectively [12-22]. However, several lines of evidence challenge this overly simplistic role for endoglin. First, an embryonic lethal phenotype is observed in mice bearing homozygous deletions [1,2]. Second, the significance of endoglin signaling to the Smad pathways during angiogenesis is not fully understood, since these canonical signal transducers can be activated without endoglin expression [25]. My preliminary findings indicate that endoglin engages other important signaling and cellular functions such as regulating Akt activation and downstream cell survival mechanisms. I have so far characterized the ability for endoglin to differentially regulate Akt activation in response to its direct ligands, TGF-2 and BMP-9. A very intriguing aspect my research proposal tries to address, therefore, is how endoglin recognizes two structurally related ligands to elicit such divergent effects on Akt activation. Another innovative aspect of my research proposal involves the plan to use in vitro model systems that incorporate many, if not all, of the separate components of the angiogenic process, such as endothelial cell proliferation, degradation of basement membrane, migration, alignment, and capillary tube formation. Several groups have employed cell proliferation and migration assays to dissect the mechanisms by which endoglin regulates angiogenesis. However, appropriate in vitro systems that comprise most of the sequential stages of angiogenesis would be favorable to monitor the effects of endoglin during angiogenic progression. Manipulating various conditions such as ligand treatment or effects of altering protein levels, would all be possible. I have begun employing such methods to study endoglin function. My preliminary findings based on capillary tube formation assay suggest that endoglin plays an important role in regulating the stability of sprouted capillary tubes, and that this dynamic process is largely governed by Akt signaling and cell survival mechanisms. How endoglin might contribute to the stability of these maturing vessels during angiogenesis is an entirely unexplored area of research, and the basis of my research plan. By employing several new in vitro angiogenesis assays including micro- carrier and co-culturing methods, the proposed work will yield significant insight into when and how endoglin exerts its effects during angiogenesis.