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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL087308-04
Application #
8011969
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Gao, Yunling
Project Start
2008-01-01
Project End
2012-12-31
Budget Start
2011-01-01
Budget End
2012-12-31
Support Year
4
Fiscal Year
2011
Total Cost
$368,929
Indirect Cost
Name
University of Missouri-Columbia
Department
Pharmacology
Type
Schools of Medicine
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
Davis, George E; Kim, Dae Joong; Meng, Chun-Xia et al. (2013) Control of vascular tube morphogenesis and maturation in 3D extracellular matrices by endothelial cells and pericytes. Methods Mol Biol 1066:17-28
Stratman, Amber N; Davis, George E (2012) Endothelial cell-pericyte interactions stimulate basement membrane matrix assembly: influence on vascular tube remodeling, maturation, and stabilization. Microsc Microanal 18:68-80
Sacharidou, Anastasia; Stratman, Amber N; Davis, George E (2012) Molecular mechanisms controlling vascular lumen formation in three-dimensional extracellular matrices. Cells Tissues Organs 195:122-43
Davis, George E (2011) Angiogenesis and Proteinases: Influence on Vascular Morphogenesis, Stabilization and Regression. Drug Discov Today Dis Models 8:13-20
Senger, Donald R; Davis, George E (2011) Angiogenesis. Cold Spring Harb Perspect Biol 3:a005090
Davis, George E; Stratman, Amber N; Sacharidou, Anastasia et al. (2011) Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting. Int Rev Cell Mol Biol 288:101-65
Stratman, Amber N; Schwindt, Amy E; Malotte, Kristine M et al. (2010) Endothelial-derived PDGF-BB and HB-EGF coordinately regulate pericyte recruitment during vasculogenic tube assembly and stabilization. Blood 116:4720-30
Davis, George E (2010) Matricryptic sites control tissue injury responses in the cardiovascular system: relationships to pattern recognition receptor regulated events. J Mol Cell Cardiol 48:454-60
Sacharidou, Anastasia; Koh, Wonshill; Stratman, Amber N et al. (2010) Endothelial lumen signaling complexes control 3D matrix-specific tubulogenesis through interdependent Cdc42- and MT1-MMP-mediated events. Blood 115:5259-69
Fisher, Kevin E; Sacharidou, Anastasia; Stratman, Amber N et al. (2009) MT1-MMP- and Cdc42-dependent signaling co-regulate cell invasion and tunnel formation in 3D collagen matrices. J Cell Sci 122:4558-69

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