This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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