Molecular Transducers Linking Shear Stress to Capillary Sprouting and Stability

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，这可能指示细胞行为的重要变化。最近，我们发现了一种以CD36下调为标志的毛细血管发芽特异性血管生成“CD36low”内皮表型。有趣的是，CD36是血小板反应蛋白-1 （TSP-1）发挥其抗血管生成活性的受体。从这些基本观察中，我们产生了一个假设，即流体剪切应力暴露的损失引发了血管生成表型的表达，而血管生成表型反过来又促进了持续发芽。为了支持这一假设，我们发现剪切应力的退出是CD36低表型的有效诱导性因子，产生的CD36表达模式与芽中的体内观察一致。此外，我们还对鉴定和测试受剪切应力调节的其他分子感兴趣，这些分子对发芽和/或毛细管稳定性至关重要。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）作为潜在的候选者。此外，我们还证实了体外结果，表明ANG-2在芽尖处高表达。总的来说，这些结果特别重要，因为到目前为止，还没有剪切应力和发芽/稳定性之间的分子联系得到机械验证。这项建议包括两个具体目标。在目标1中，我们将确定将“毛细血管发芽”剪切应力方案应用于内皮细胞是否会引发在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“剪切撤回”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胁迫的调节一致。这项分析将包括CD36、ANG-1、ANG-2和VEGF-C，这些已经在初步研究中涉及到。在Aim #2中，我们将通过改变CD36、ANG-1、ANG-2和/或VEGF-C的表达，确定用相同的“毛细血管发芽”剪切应力方案进行预处理的内皮细胞是否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著改变血管生成功能，那么剪切应力与毛细血管发芽/微血管稳定性之间的分子联系将首次从机制上分离出来。

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.