Molecular Transducers Linking Shear Stress to Capillary Sprouting and Stability

将剪切应力与毛细管萌芽和稳定性联系起来的分子传感器

• 批准号: 8109956
• 负责人: Richard J. Price
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8109956
• 关键词: Affect; Amplifiers; Angiopoietin-1; Apoptosis; Area; Behavior; Biochemical; Biological Assay; Blood Vessels; Blood capillaries; Blood flow; CD36 gene; Cell Proliferation; Cell physiology; Cells; Custom; Data; Devices; Down-Regulation; Elements; Endothelial Cells; Excision; Exhibits; Exposure to; Feedback; In Vitro; Inflammation; Injury; Invaded; Legal patent; Link; Liquid substance; Membrane Glycoproteins; Messenger RNA; Microscopic; Modeling; Molecular; Organ; Paper; Pattern; Permeability; Phase; Phenotype; Positioning Attribute; Process; Proliferating; Protocols documentation; Rattus; Regulation; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Signal Pathway; Signal Transduction; Simulate; Stimulus; Surface; Testing; Text; Thrombospondin 1; Time; Tissues; Transducers; United States National Institutes of Health; Up-Regulation; Vascular Endothelial Growth Factor C; Vascular Endothelial Growth Factors; Western Blotting; Withdrawal; Wound Healing; angiogenesis; base; capillary; cell behavior; cell motility; conditioning; design; in vivo; interest; migration; protein expression; public health relevance; receptor; restoration; shear stress

DESCRIPTION (provided by applicant): During angiogenesis, subsets of endothelial cells exhibit unique phenotypes that may be indicative of important changes in cell behavior. Recently, we identified a capillary sprout-specific angiogenic "CD36low" endothelial phenotype that is marked by the downregulation of CD36. Interestingly, CD36 is the receptor through which Thrombospondin-1 (TSP-1) exerts its anti-angiogenic activity. From these basic observations, we generated the hypothesis that the loss of exposure to fluid shear stress elicits the expression of an angiogenic phenotype which, in turn, facilitates continued sprouting. In support of this hypothesis, we have found that the withdrawal of shear stress is a potent inducer of the CD36low phenotype, yielding CD36 expression patterns that are consistent with in vivo observations in sprouts. In addition, we are also interested in identifying and testing other molecules that are regulated by shear stress and critical for sprouting and/or capillary stability. To this end, we have identified angiopoietin-1 (ANG-1), ANG-2, and vascular endothelial growth factor-C (VEGF-C) as potential candidates. Moreover, we have confirmed the in vitro results by showing that ANG-2 is highly expressed at sprout tips. Overall, these results are particularly significant because, to date, no molecular linkages between shear stress and sprouting/stability have been mechanistically verified. This proposal consists of two specific aims. In Aim #1, we will determine whether the application of a "capillary sprouting" shear stress protocol to endothelial cells elicits an angiogenic phenotype that is recapitulated in vivo. Here, we will use a custom RT-PCR array to characterize the "shear-withdrawn" sprouting phenotype and determine whether the in vivo expression patterns of selected phenotypic markers are consistent with regulation by shear stress. This analysis will include CD36, ANG-1, ANG-2, and VEGF-C, which have already been implicated in preliminary studies. In Aim #2, we will determine whether endothelial cells that are pre-conditioned with the same "capillary sprouting" shear stress protocol exhibit enhanced angiogenic function (i.e. proliferation, migration, and barrier function) through changes in the expression of CD36, ANG-1, ANG-2, and/or VEGF-C. If selectively blocking and/or enhancing one or more of these candidate molecules significantly alters angiogenic function, a molecular linkage between shear stress and capillary sprouting/microvessel stability will have been mechanistically isolated for the first time. PUBLIC HEALTH RELEVANCE: During inflammation and tissue injury, new microscopic blood vessels grow by sprouting from existing blood vessels. This process is essential for building functional blood vessel networks in the repaired tissue, and we hypothesize that the withdrawal of exposure to shear stress, which is generated by blood flow, enhances the ability of endothelial cells to make sprouts. In this proposal, we will determine how the removal of shear stress from endothelial cells affects both the expression of proteins associate with sprouting, as well as endothelial cell proliferation, migration, and permeability. In addition, by blocking the function of molecules that are shown to be altered with the removal of shear stress, we will determine which molecules are responsible for eliciting sprouting behaviors.

描述（由申请人提供）：在血管生成过程中，内皮细胞亚群表现出独特的表型，这可能表明细胞行为的重要变化。最近，我们发现了一种毛细血管芽特异性血管生成“CD36low”内皮表型，其特征是 CD36 下调。有趣的是，CD36 是 Thrombospondin-1 (TSP-1) 发挥其抗血管生成活性的受体。根据这些基本观察，我们提出了这样的假设：失去流体剪切应力的暴露会引发血管生成表型的表达，进而促进持续发芽。为了支持这一假设，我们发现剪切应力的撤消是 CD36low 表型的有效诱导剂，产生的 CD36 表达模式与豆芽中的体内观察结果一致。此外，我们还对识别和测试受剪切应力调节并对发芽和/或毛细管稳定性至关重要的其他分子感兴趣。为此，我们确定了血管生成素-1 (ANG-1)、ANG-2 和血管内皮生长因子-C (VEGF-C) 作为潜在候选药物。此外，我们通过显示 ANG-2 在芽尖高表达来证实体外结果。总的来说，这些结果特别重要，因为迄今为止，剪切应力和发芽/稳定性之间的分子联系尚未得到机械验证。 该提案包含两个具体目标。在目标#1中，我们将确定对内皮细胞应用“毛细血管出芽”剪切应力方案是否会引发体内重现的血管生成表型。在这里，我们将使用定制的 RT-PCR 阵列来表征“剪切撤退”的发芽表型，并确定所选表型标记的体内表达模式是否与剪切应力的调节一致。该分析将包括 CD36、ANG-1、ANG-2 和 VEGF-C，这些已在初步研究中涉及。在目标#2中，我们将确定用相同的“毛细血管出芽”剪切应力方案预处理的内皮细胞是否通过CD36、ANG-1、ANG-2和/或VEGF-C表达的变化表现出增强的血管生成功能（即增殖、迁移和屏障功能）。如果选择性阻断和/或增强这些候选分子中的一个或多个显着改变血管生成功能，则剪切应力和毛细血管出芽/微血管稳定性之间的分子联系将首次被机械分离。 公共卫生相关性：在炎症和组织损伤期间，新的微观血管会从现有血管中生长出来。这个过程对于在修复的组织中构建功能性血管网络至关重要，我们假设，摆脱血流产生的剪切应力可以增强内皮细胞发芽的能力。在本提案中，我们将确定去除内皮细胞的剪切应力如何影响与出芽相关的蛋白质的表达，以及内皮细胞的增殖、迁移和通透性。此外，通过阻断随着剪切应力的消除而改变的分子的功能，我们将确定哪些分子负责引发发芽行为。