MATRIX METALLOPROTEINASE IN VASCULOGENESIS & ANGIOGENESIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们研究的广泛和长期目标是开发分子工具，使我们能够促进或抑制体内血管生成。然后，这些工具可以用于治疗人类疾病和血管组织工程。来自我们实验室和其他研究人员的研究强调ADAM-17是一个关键的，但目前被低估的，在血管生成调节的几个水平上的参与者。在初步研究中，我们已经表明，下调HUVECs中ADAM-17的表达对其在血管生成模型系统中的行为具有深远的影响。特别是，HUVEC需要ADAM-17在Matrigel上形成网络，响应VEGF而侵袭Matrigel，并表达激活的ERK和MMP-2以响应VEGF。这些联合观察支持了我们的假设，即ADAM-17是血管生成的中央调节因子，因为：1)它被血管内皮生长因子激活，2)它裂解多种底物，3)它与其他蛋白质形成复合体，4)它对内皮细胞、辅助细胞及其相互作用的整体影响。与大多数通过去除前肽而激活的金属蛋白酶不同，缺乏前肽的ADAM-17在酶活性上是不活跃的。由于我们认为ADAM-17的促血管生成功能中的大多数(如果不是全部)都需要VEGF的存在，在我们的第一个特定目的中，我们检验了这样的假设，即VEGF激活ADAM-17，并且这种激活是通过分子的磷酸化或酶切割的变化而发生的。我们将进一步研究ADAM-17在HUVEC中的亚细胞分布是否受到VEGF信号的影响。在我们的第二个特定目标中，我们将研究ADAM-17是否通过切割已知的底物如ErbB配体、细胞-细胞黏附分子(选择素、VCAM、ICAM、钙粘附素)在血管生成中发挥作用。我们还将尝试使用非活性催化结构域捕获技术来识别新的底物。ADAM-17的促血管生成功能与其催化活性无关的可能性也将在使用非催化活性的ADAM-17的实验中进行评估。在特定的目标三，我们将使用免疫共沉淀来研究ADAM-17由于与细胞质蛋白或与其他细胞表面蛋白的分子间相互作用而被激活的可能性。在第四个具体目标中，我们将使用微阵列进行实验，以确定当HUVEC培养中ADAM-17表达被下调时，其表达发生变化的mRNA转录本。虽然这些研究可能会揭示更多属于ADAM-17配体或底物的蛋白质，但更有可能的是，这些研究将揭示具有各种不同、意想不到的功能的蛋白质，其表达受到ADAM-17的调控。当这些蛋白质被确定后，将制定计划来表征它们在血管生成中的作用。关于ADAM-17的激活、ADAM-17的底物、ADAM-17的配体以及由ADAM-17调控表达的蛋白质的拟议研究结果应该会为我们提供丰富的新数据，这些数据将被用于制定R01在NIH的应用。特别是，这些数据应该为治疗提供各种新的靶点，根据临床环境的需要具体增强或抑制血管生成，并应该建议改进制造组织工程血管的方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The broad, long-term objective of our studies is to develop molecular tools that will allow us to enhance or inhibit angiogenesis in vivo. These tools can then be used in the treatment of human diseases and in the tissue engineering of blood vessels. Studies from our laboratory and others highlight ADAM-17 as a key, yet currently underappreciated, player at several levels in the regulation of angiogenesis. In Preliminary Studies, we have shown that knocking down ADAM-17 expression in HUVECs has profound effects on their behavior in model systems for angiogenesis. In particular, HUVECs require ADAM-17 to form networks on Matrigel, to invade Matrigel in response to VEGF, and to express activated ERK and MMP-2 in response to VEGF. These combined observations support our hypothesis that ADAM-17 is a central regulator of angiogenesis due to: 1) its activation by VEGF, 2) its cleavage of a variety of substrates, 3) its formation of complexes with other proteins, and 4) its global effects on ECs, accessory cells, and their interactions. Unlike most metalloproteinases that are activated by removal of a propeptide, ADAM-17 lacking a propeptide is enzymatically inactive. Because most, if not all, of the pro-angiogenic functions that we have attributed to ADAM-17 require the presence of VEGF, in our first specific aim we test the hypothesis that VEGF activates ADAM-17 and that this activation occurs via change in the phosphorylation or enzymatic cleavage of the molecule Further we will study whether the subcellular distribution of ADAM-17 in HUVEC is affected by VEGF signaling. In our second specific aim we will investigate whether ADAM-17 plays a role in angiogenesis by cleaving known substrates such as ErbB ligands, cell-cell adhesion molecules (selectins, VCAM, ICAM, cadherins). We will also attempt to identify novel substrates using the inactive catalytic domain capture technique. The possibility that the pro-angiogenic functions of ADAM-17 are independent of its catalytic activity will also be evaluated in experiments using catalytically-inactive ADAM-17. In specific aim three we will use co-immunoprecipitation to study the possibility that ADAM-17 becomes activated due to intermolecular interactions with cytoplasmic proteins or with other cell surface proteins. In the fourth specific aim we will conduct experiments using microarrays to identify mRNA transcripts whose expression is altered when ADAM-17 expression is knocked down in HUVEC cultures. While these studies may reveal additional proteins that are ADAM-17 ligands or substrates, it is more likely that these studies will reveal proteins with a variety of different, unexpected functions whose expression is regulated by ADAM-17. When such proteins are identified, plans will be made to characterize their roles in angiogenesis. The results of the proposed studies on the activation of ADAM-17, substrates for ADAM-17, ligands for ADAM-17, and proteins whose expression is regulated by ADAM-17 should provide us with a wealth of novel data that will be used to craft an R01 application to NIH. In particular, these data should suggest a variety of novel targets for treatments that will specifically enhance or inhibit angiogenesis as desired in clinical settings and should suggest improved approaches for producing tissue engineered blood vessels.