Endoglin Regulates Endothelial Survival and Capillary Tube Stability

内皮糖蛋白调节内皮细胞存活和毛细管稳定性

• 批准号: 8111481
• 负责人: Nam Y Lee
• 金额: $ 8.94万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-20 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8111481
• 关键词: Address; Affinity; Angiogenesis Inhibitors; Area; Basement membrane; Biological; Biological Assay; Biological Models; Biology; Blood Vessels; Blood capillaries; Caspase; Cell Proliferation; Cell Survival; Cell physiology; Coculture Techniques; Complex; Data; Defect; ENG gene; Embryo; Endoglin; Endothelial Cells; Equilibrium; Family; Hereditary hemorrhagic telangiectasia; Human; In Vitro; Inflammation; Ligands; Mediating; Methods; Migration Assay; Molecular; Monitor; Morphogenesis; Mus; Mutation; Outcome; Pathway interactions; Patients; Phase; Phenotype; Play; Process; Proteins; Regulation; Reporting; Research; Research Proposals; Role; Signal Pathway; Signal Transduction; Staging; Structure; System; TGF-beta type I receptor; Testing; Transducers; Transforming Growth Factors; Tube; Vascular Diseases; Work; angiogenesis; base; bone morphogenetic protein 9; capillary; cell behavior; improved; in vitro Model; innovation; insight; migration; novel; protein transport; receptor; response; scaffold; tumor

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.

描述（由申请人提供）：具体目标内皮细胞中的转化生长因子（TGF-2）超家族信号传导调节血管生成和血管形态发生的重要组成部分，包括增殖和毛细管形成。 TGF-2 超家族配体通过内皮细胞特异性 TGF-2 受体复合物 ALK1（I 型受体）和内皮糖蛋白（共受体）以及普遍存在的 I 型 TGF-2 受体 ALK5 发挥调节作用，分别激活经典的 Smad 1/5/8 和 Smad 2/3 通路。 TGF-2 配体还通过非 Smad 途径（例如 MAPK 和 PI3K/Akt）发出信号，但其潜在机制仍不清楚。内皮糖蛋白和 ALK1 在内皮细胞中 TGF-2 信号转导中的关键作用是由它们的突变导致的，这些突变导致人类血管疾病、遗传性出血性毛细血管扩张症（HHT1 和 2）、当内皮糖蛋白或 ALK1 被靶向删除小鼠时血管生成缺陷导致的胚胎致死表型，以及炎症和炎症期间内皮糖蛋白的表达升高。 肿瘤诱导的血管生成。虽然内皮糖蛋白的重要生物学作用已经确定，但血管生物学中内皮糖蛋白功能的分子基础仍然知之甚少。这里采用体外血管生成测定来精确研究内皮糖蛋白何时以及如何调节内皮毛细血管萌芽和管形成。内皮糖蛋白无效和野生型内皮细胞的比较表明，内皮糖蛋白响应其生理相关的高亲和力配体TGF-2和BMP-9，差异性地调节毛细血管芽和管的稳定性。具体而言，TGF-2 主要通过抑制内皮糖蛋白依赖性 Akt 信号传导导致毛细血管芽和管结构退化。相反，内皮糖蛋白增强 Akt 信号传导以响应 BMP-9，从而促进毛细血管稳定性。这些结果归因于内皮糖蛋白和支架/​​运输蛋白 GIPC 之间的关联，因为破坏它们的相互作用会消除这种内皮糖蛋白依赖性效应。鉴于我最近报道了通过 GIPC 和内皮糖蛋白增强 Smad 1/5/8 信号传导，Akt 和内皮糖蛋白依赖性 Smad 1/5/8 信号传导之间存在潜在的串扰，我建议对此进行研究。最后，我发现了内源性内皮糖蛋白和 Akt 之间的一种新的相互作用，这一发现可能会产生内皮糖蛋白生物学的新方面。基于这些初步数据，我提出以下假设：Endoglin 与 GIPC 结合，通过 BMP-9 依赖性 Akt 激活来稳定内皮毛细血管，同时通过 TGF-2 依赖性 Akt 信号传导和细胞存活机制的抑制来不稳定毛细血管，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。 公共健康相关性：我的申请的一个明显创新的方面是定义了内皮糖蛋白在改变内皮细胞行为中的新信号作用。目前的范式将内皮糖蛋白描述为辅助 TGF-2 共受体，调节两个相反信号通路 Smad1/5/8 和 Smad2/3 的平衡，分别调用促血管生成反应或抗血管生成反应 [12-22]。然而，一些证据对内皮糖蛋白这种过于简单化的作用提出了质疑。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，血管生成过程中内皮糖蛋白信号转导对 Smad 通路的重要性尚未完全了解，因为这些经典信号转导器可以在没有内皮糖蛋白表达的情况下被激活 [25]。我的初步研究结果表明内皮糖蛋白参与其他重要的信号传导和细胞功能，例如调节 Akt 激活和下游细胞存活机制。到目前为止，我已经表征了内皮糖蛋白响应其直接配体 TGF-2 和 BMP-9 差异调节 Akt 激活的能力。因此，我的研究计划试图解决的一个非常有趣的方面是内皮糖蛋白如何识别两个结构相关的配体以引发对 Akt 激活的不同影响。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，该系统包含血管生成过程的许多（如果不是全部）独立组成部分，例如内皮细胞增殖、基底膜降解、迁移、排列和毛细管形成。几个小组已经利用细胞增殖和迁移测定来剖析内皮糖蛋白调节血管生成的机制。然而，包含血管生成的大部分连续阶段的适当的体外系统将有利于监测血管生成进展期间内皮糖蛋白的作用。操纵各种条件，例如配体处理或改变蛋白质水平的效果，都是可能的。我已经开始采用这样的方法来研究内皮糖蛋白功能。我基于毛细管形成实验的初步发现表明，内皮糖蛋白在调节发芽毛细管的稳定性方面发挥着重要作用，并且这一动态过程很大程度上受 Akt 信号传导和细胞存活机制的控制。内皮糖蛋白如何在血管生成过程中促进这些成熟血管的稳定性是一个完全未经探索的研究领域，也是我的研究计划的基础。通过采用几种新的体外血管生成测定，包括微载体和共培养方法，所提出的工作将深入了解内皮糖蛋白在血管生成过程中何时以及如何发挥其作用。

项目成果

Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation.

• DOI: 10.1016/j.bbrc.2012.06.163
• 发表时间: 2012-08-03
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: Pan CC;Bloodworth JC;Mythreye K;Lee NY
• 通讯作者: Lee NY