Pericyte Proteinase Inhibitors and EC Tube Stabilization

描述（由申请人提供）：这项新申请解决了血管生物学中的一个重要问题，即新形成的内皮细胞（EC）内衬管是如何稳定的。我们已经在体外开发了这一过程的优秀模型，并确定了这些事件所需的关键机制和分子。ec衍生的蛋白酶，如MMP-1，能够在小管形态形成过程中降解3D胶原基质，从而使小管不稳定并诱导退化。我们假设EC支持细胞（如周细胞）存在蛋白酶抑制剂，如TIMP-3和RECK，它们在血管生成过程中阻止EC降解其悬浮的细胞外基质环境的自然倾向。这些周细胞衍生的抑制剂可能代表了对新形成的管的稳定至关重要的新分子。初步数据表明，TIMP-3的独特之处在于它可以完全抑制EC管的形态发生，并稳定EC衬管。此外，TIMP-3在周细胞的原代培养中大量表达，而在内皮细胞中不表达。血管平滑肌细胞（也表达TIMP-3）与ECs在MMP-1依赖性管回归的3D模型中通过干扰MMP-1前酶激活完全阻断了这一过程。我们将利用平衡的实验方法来确定周细胞来源的蛋白酶抑制剂在体外和体内EC管稳定中的作用（使用重组腺病毒基因传递和TIMP-3敲除小鼠）。在血管生成和血管发育过程中，周细胞如何稳定EC管的机制尚不清楚，该建议将直接验证蛋白酶抑制剂是调节这些事件的关键分子的新假设。

DESCRIPTION (provided by applicant): This new application addresses an important question in vascular biology which concerns how newly formed endothelial cell (EC)-lined tubes are stabilized. We have developed excellent models of this process in vitro and have identified key mechanisms and molecules, which are required for these events. EC-derived proteinases such as MMP-1 are capable of degrading 3D collagen matrices during tubular morphogenesis, which destabilizes tubes and induces regression. We hypothesize that EC supporting cells such as pericytes present proteinase inhibitors such as TIMP-3 and RECK which blocks the natural tendency of ECs during angiogenesis to degrade the extracellular matrix environment in which they are suspended. These pericyte-derived inhibitors likely represent new molecules that are critical to the stabilization of newly formed tubes. Preliminary data shows that TIMP-3 is unique in that it can completely inhibit EC tubular morphogenesis as well as stabilize EC-lined tubes. Furthermore, TIMP-3 is heavily expressed by primary cultures of pericytes and is not expressed by ECs. Inclusion of vascular smooth muscle cells (also express TIMP-3) with ECs in 3D models of MMP-1-dependent tube regression completely blocks the process by interfering with MMP-1 proenzyme activation. We will utilize a balanced experimental approach to determine the role of pericyte-derived proteinase inhibitors in EC tube stabilization in vitro and in vivo (using recombinant adenoviral gene delivery and TIMP-3 knockout mice). The mechanisms involved in how pericytes stabilize EC tubes during angiogenesis and vascular development remain unknown and this proposal will directly test the novel hypothesis that proteinase inhibitors are key molecules regulating these events. The specific aims of this application are; Aim #1. To investigate the primary mechanisms and molecules (i.e. proteinase inhibitors and growth factors) which regulate the ability of pericytes/vascular smooth muscle cells to induce capillary tube stabilization in vitro and in vivo. Aim #2. To investigate the molecular mechanism by which TIMP-3 regulates capillary tube stabilization in vitro and in vivo through complete inhibition of EC invasion and lumen development during tubular morphogenesis in three-dimensional matrices. Aim #3. To investigate the ability of human matrix metalloproteinase-1 (MMP-1) as well as mouse interstitial collagenases (e.g. MMP-13) to directly control capillary tube regression events in vitro and in vivo.