MATRIX METALLOPROTEINASE IN VASCULOGENESIS & ANGIOGENESIS

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们研究的广泛的长期目标是开发分子工具，使我们能够增强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的组织工程。来自我们实验室和其他人的研究强调了ADAM-17作为一个关键的，但目前被低估的，在血管生成调控的几个层面上的球员。在初步研究中，我们已经表明，敲低HUVEC中的ADAM-17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侵入Matrigel，并响应VEGF表达活化的ERK和MMP-2。这些组合的观察结果支持我们的假设，即ADAM-17是血管生成的中心调节因子，这是由于：1）其被VEGF激活，2）其切割多种底物，3）其与其他蛋白质形成复合物，以及4）其对EC、辅助细胞及其相互作用的全局影响。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是无酶活性的。因为我们归因于ADAM-17的大多数（如果不是全部）促血管生成功能需要VEGF的存在，所以在我们的第一个具体目标中，我们测试了VEGF激活ADAM-17的假设，并且这种激活通过分子的磷酸化或酶裂解的变化发生。在我们的第二个具体目标中，我们将研究ADAM-17是否通过切割已知底物如ErbB配体、细胞-细胞粘附分子（选择素、VCAM、ICAM、钙粘蛋白）在血管生成中起作用。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的促血管生成功能不依赖于其催化活性的可能性也将在使用无催化活性的ADAM-17的实验中进行评估。在具体目标三中，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鉴定当在HUVEC培养物中敲低ADAM-17表达时其表达改变的mRNA转录物。虽然这些研究可能揭示了其他的蛋白质是ADAM-17配体或底物，但这些研究更有可能揭示具有各种不同的、意想不到的功能的蛋白质，其表达受ADAM-17调节。当这些蛋白质被鉴定出来后，将制定计划来描述它们在血管生成中的作用。对ADAM-17的激活、ADAM-17的底物、ADAM-17的配体以及其表达受ADAM-17调节的蛋白质的拟议研究的结果应该为我们提供大量的新数据，这些数据将用于将R 01应用于NIH。特别是，这些数据应该提出各种新的治疗目标，将具体地增强或抑制血管生成，如在临床环境中所需的，并应该提出改进的方法，用于生产组织工程血管。

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.