Pericyte Proteinase Inhibitors and EC Tube Stabilization

描述(申请人提供)：这项新的申请解决了血管生物学中的一个重要问题，该问题涉及到新形成的内皮细胞(EC)衬里的管是如何稳定的。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。基质金属蛋白酶-1等EC衍生的蛋白水解酶能够在肾小管形态形成过程中降解3D胶原基质，从而破坏肾小管的稳定性，导致肾小管退行性变。我们假设，支持EC的细胞，如周细胞，提供了TIMP-3和RECK等蛋白酶抑制剂，从而阻断了ECs在血管生成过程中降解其悬浮所在的细胞外基质环境的自然趋势。这些周细胞衍生的抑制物可能代表了对新形成的管子的稳定至关重要的新分子。初步数据表明，TIMP-3是独一无二的，它可以完全抑制EC管的形态发生，并稳定EC内衬的管。此外，TIMP-3在原代培养的周细胞中大量表达，而在内皮细胞中不表达。将血管平滑肌细胞(也表达TIMP-3)与内皮细胞一起包含在依赖于基质金属蛋白酶-1的小管回归的3D模型中，通过干扰基质金属蛋白酶-1酶原的激活来完全阻断这一过程。我们将利用一种平衡的实验方法，在体外和体内(使用重组腺病毒基因传递和TIMP-3基因敲除小鼠)确定周细胞衍生的蛋白水解酶抑制物在EC管稳定中的作用。周细胞在血管生成和血管发育过程中如何稳定EC管的机制尚不清楚，这一提议将直接检验蛋白酶抑制剂是调节这些事件的关键分子这一新假说。 本申请的具体目的是： 目的：1.在体内外研究调控周细胞/血管平滑肌细胞诱导毛细血管稳定的主要机制和分子(即蛋白酶抑制剂和生长因子)。 目的#2.研究TIMP-3通过完全抑制三维基质中内皮细胞的侵袭和管腔发育，在体内外调节毛细血管的稳定性的分子机制。 目的#3.研究人基质金属蛋白酶-1(MMP1)和小鼠间质胶原酶(如MMP13)在体内外直接控制毛细血管退行性变的能力。

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.