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.
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