Molecular Control of EC Lumen Formation by MT1-MMP

MT1-MMP 对 EC 腔形成的分子控制

• 批准号: 7373336
• 负责人: George E Davis
• 金额: $ 37.36万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7373336
• 关键词: Address; Adherens Junction; Attention; Basement membrane; Biological Assay; Biological Models; Biology; Blood; Blood Vessels; Cell Line; Cell surface; Cells; Chemicals; Chimera organism; Collagen; Data; Development; Diabetes Mellitus; Endopeptidases; Endothelial Cells; Environment; Equilibrium; Event; Extracellular Matrix; Facility Construction Funding Category; Fibrin; Fluorescence Microscopy; GM 6001; Green Fluorescent Proteins; Human; Immunoprecipitation; In Vitro; Integrin Binding; Integrins; Investigation; Laboratories; Link; Localized; Lymphatic; Lymphatic Endothelial Cells; MMP14 gene; Malignant Neoplasms; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Membrane; Metalloproteases; Modeling; Molecular; Morphogenesis; Movement; Pathogenesis; Pathway interactions; Peptide Hydrolases; Pericytes; Peripheral Vascular Diseases; Personal Satisfaction; Phenotype; Play; Process; Proteins; Proteolysis; Proteomics; Recombinants; Role; Series; Signal Transduction; Small Interfering RNA; Smooth Muscle Myocytes; Structure; Time; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-3; Tissue Inhibitor of Metalloproteinases; Tube; Tubular formation; Vacuole; Work; Zebrafish; angiogenesis; base; cell motility; cell type; human MMP14 protein; human disease; in vivo; in vivo Model; inhibitor/antagonist; knock-down; prevent; protein expression; response; rho GTP-Binding Proteins; tool; vasculogenesis

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.

描述（由申请人提供）：此修订的应用程序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）在3D细胞外基质环境中如何形成流明的分子机制。我们已经在体外开发了这一过程的出色模型，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EC腔形成的新调节剂，该组织是EC衍生的蛋白酶，膜型金属蛋白酶-1（MT1-MMP）。 EC流明的形成是需要催化胶原蛋白矩阵局部细胞表面蛋白水解的能力所必需的。这种局部蛋白水解产生了一个“血管引导隧道”网络，该网络在3D胶原矩阵的形态学过程中引导EC迁移。使用化学（GM6001）或TIMP-2和TIMP-3等蛋白质抑制剂对MT1-MMP的阻断会导致EC腔形成完全干扰（通过阻断引导隧道形成）和管状形态发生。延时分析表明，TIMP-2和TIMP-3而不是TIMP-1可以防止EC形成腔内结构，而是发出了小的精细过程。与外源添加的MT1-MMP抑制剂相比，使用siRNA治疗对EC中MT1-MMP的抑制显着阻断了腔的形成，从而导致表型相同。我们还提供了初步数据，表明人淋巴EC需要膜MMP也形成腔内结构。我们提出了一种平衡的实验方法，以确定MT1-MMP及其相关的调节分子如何在体外和体内控制血液与淋巴EC流明的过程。所研究的分子机制将揭示这些事件的关键信息，包括为什么EC，而不是通过这种MT1-MMP依赖性机制形成流明的周围细胞，而不是血管周围的细胞。该应用程序的具体目的是；目标＃1。确定MT1-MMP在血管中与淋巴管脉管系统中内皮细胞流腔形成的分子控制中的作用，体外和体内。目标＃2。识别和表征MT1-MMP相关和调节分子的功能，这些分子控制其诱导内皮细胞腔形成事件的能力。目标＃3。为了确定人类内皮细胞中的MT1-MMP表达如何通过构造“血管引导隧道”来导致腔形成，从而调节血管血管和淋巴管血管内皮管的形态发生。