Molecular Control of EC Lumen Formation by MT1-MMP

描述（由申请人提供）：本修订申请解决了血管生物学中的一个重要问题，该问题涉及人内皮细胞（EC）如何在3D细胞外基质环境中形成管腔的分子机制。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。在初步研究中，我们已经确定了一个新的调节EC管腔形成，这是EC衍生的蛋白酶，膜型金属蛋白酶-1（MT 1-MMP）。通过其催化胶原基质的局部细胞表面定向蛋白水解的能力，EC管腔形成需要它。这种局部蛋白水解产生了一个“血管引导隧道”网络，在3D胶原基质中的形态发生过程中引导EC迁移。使用化学（GM 6001）或蛋白质抑制剂如TIMP-2和TIMP-3阻断MT 1-MMP导致EC管腔形成（通过阻断引导通道形成）和管状形态发生的完全干扰。时间推移分析显示，TIMP-2和TIMP-3，但不是TIMP-1，阻止EC形成内腔结构，而是它们发出小的精细过程。使用siRNA处理抑制EC中的MT 1-MMP显著阻断管腔形成，导致与外源性添加的MT 1-MMP抑制剂相同的表型。我们还提出了初步的数据显示，人类淋巴管内皮细胞需要膜基质金属蛋白酶形成管腔结构以及。我们提出了一个平衡的实验方法，以确定如何MT 1-MMP及其相关的调节分子控制的过程中，血液与淋巴EC管腔形成在体外和体内。研究的分子机制将揭示这些事件背后的关键信息，包括为什么EC而不是血管周围细胞如周细胞通过这种MT 1-MMP依赖性机制形成管腔。本申请的具体目标是：目标#1。确定MT 1-MMP在体外和体内血管系统与淋巴管系统中内皮细胞管腔形成的分子控制中的作用。目标2。鉴定和表征MT 1-MMP相关和调节分子的功能，这些分子控制其诱导内皮细胞管腔形成事件的能力。目标3。为了确定MT 1-MMP在人内皮细胞（而不是在其他血管周围细胞）中的表达如何通过构建调节血液血管和淋巴管内皮管形态发生的“血管引导隧道”导致管腔形成。

DESCRIPTION (provided by applicant): This revised application addresses an important question in vascular biology which concerns the molecular mechanisms that underlie how human endothelial cells (ECs) form lumens in 3D extracellular matrix environments. We have developed excellent models of this process in vitro and have identified key mechanisms and molecules that are required for these events. In preliminary studies, we have identified a new regulator of EC lumen formation that is the EC-derived proteinase, membrane-type metalloproteinase-1 (MT1-MMP). It is required for EC lumen formation through its ability to catalyze local cell surface directed proteolysis of collagen matrices. This localized proteolysis generates a network of "vascular guidance tunnels" which direct EC migration during the morphogenic process in 3D collagen matrices. Blockade of MT1-MMP using chemical (GM6001) or protein inhibitors such as TIMP-2 and TIMP-3 results in complete interference of EC lumen formation (by blocking guidance tunnel formation) and tubular morphogenesis. Time-lapse analysis reveals that TIMP-2 and TIMP-3, but not TIMP-1, prevents ECs from forming lumenal structures but instead they send out small fine processes. Suppression of MT1-MMP in ECs using siRNA treatment markedly blocks lumen formation resulting in an identical phenotype compared to exogenously added MT1-MMP inhibitors. We also present preliminary data showing that human lymphatic ECs require membrane MMPs to form lumenal structures as well. We propose a balanced experimental approach to determine how MT1-MMP and its associated regulatory molecules control the process of blood versus lymphatic EC lumen formation in vitro and in vivo. The molecular mechanisms investigated will reveal critical information underlying these events including why ECs, and not perivascular cells such as pericytes, form lumens through this MT1-MMP-dependent mechanism. The specific aims of this application are; Aim #1. To determine the role of MT1-MMP in the molecular control of endothelial cell lumen formation in blood vasculature versus lymphatic vasculature in vitro and in vivo. Aim #2. To identify and characterize the function of MT1-MMP-associated and regulatory molecules that control its ability to induce endothelial cell lumen formation events. Aim #3. To determine how MT1-MMP expression in human endothelial cells (and not in other perivascular cells) leads to lumen formation through the construction of "vascular guidance tunnels" which regulate blood vascular and lymphatic vascular endothelial tube morphogenesis.