Molecular Control of EC Lumen Formation by MT1-MMP

描述(申请人提供)：这项修订后的申请解决了血管生物学中的一个重要问题，该问题涉及人类内皮细胞(ECs)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，我们已经发现了一种新的EC管腔形成的调节因子，即EC衍生的蛋白酶，膜型金属蛋白酶-1(MT1-MMPs)。它通过催化局部细胞表面直接蛋白分解胶原基质的能力，是形成EC腔所必需的。这种局部的蛋白分解产生了一个“血管引导隧道”网络，在3D胶原基质的形态形成过程中引导EC迁移。使用化学药物(GM6001)或蛋白质抑制剂如TIMP-2和TIMP-3阻断MT1-MMP会导致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，而不是TIMP-1，阻止内皮细胞形成管腔结构，但它们发出细小的细小突起。使用siRNA处理抑制内皮细胞中的MT1-MMP显著阻止管腔的形成，导致与外源性添加的MT1-MMP抑制剂相比，产生相同的表型。我们还提供了初步数据显示，人类淋巴管内皮细胞也需要膜基质金属蛋白酶来形成管腔结构。我们提出了一种平衡的实验方法来确定MT1-MMP及其相关的调节分子如何在体外和体内控制血液和淋巴管EC管腔的形成过程。所研究的分子机制将揭示这些事件背后的关键信息，包括为什么内皮细胞，而不是血管周围细胞，如周细胞，通过这种MT1-基质金属蛋白酶依赖的机制形成管腔。本应用的具体目的是：目的1.确定MT1-MMP体内外在血管和淋巴管内皮细胞腔形成的分子调控中的作用。目的#2.鉴定和表征MT1-基质金属蛋白酶相关和调控分子的功能，这些分子控制其诱导内皮细胞管腔形成事件的能力。目的#3.通过构建调节血管和淋巴管内皮细胞形态的“血管引导通道”，确定MT1-MMPs在人内皮细胞(而不是其他血管周围细胞)中的表达如何导致管腔的形成。

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.