Endothelial cell lumen and vascular guidance tunnel formation requires MT1-MMP–dependent proteolysis in 3-dimensional collagen matrices

Abstract

Here we show that endothelial cells (EC) require matrix type 1-metalloproteinase (MT1-MMP) for the formation of lumens and tube networks in 3-dimensional (3D) collagen matrices. A fundamental consequence of EC lumen formation is the generation of vascular guidance tunnels within collagen matrices through an MT1-MMP-dependent proteolytic process. Vascular guidance tunnels represent a conduit for EC motility within these spaces (a newly remodeled 2D matrix surface) to both assemble and remodel tube structures. Interestingly, it appears that twice as many tunnel spaces are created than are occupied by tube networks after several days of culture. After tunnel formation, these spaces represent a 2D migratory surface within 3D collagen matrices allowing for EC migration in an MMPindependent fashion. Blockade of EC lumenogenesis using inhibitors that interfere with the process (eg, integrin, MMP, PKC, Src) completely abrogates the formation of vascular guidance tunnels. Thus, the MT1-MMP-dependent proteolytic process that creates tunnel spaces is directly and functionally coupled to the signaling mechanisms required for EC lumen and tube network formation. In summary, a fundamental and previously unrecognized purpose of EC tube morphogenesis is to create networks of matrix conduits that are necessary for EC migration and tube remodeling events critical to blood vessel assembly. (Blood. 2009;114: 237-247) IntroductionMuch progress has occurred in our understanding of the molecular events controlling the processes underlying vascularization of tissues in the context of development and disease. [1][2][3][4][5][6][7] Work that is receiving increasing attention focuses on identifying specific steps required for vascular morphogenesis, including those involving endothelial cell (EC) lumen formation. [8][9][10][11][12] In addition to the identification of specific molecules required for these events, it is important to determine how different cell types such as endothelial cells, pericytes, and vascular smooth muscle cells interact and assemble to form the different characteristic blood vessel types. 1,6,13,14 Recent work from our laboratory reveals that ECs form lumens in 3-dimensional (3D) collagen matrices through a signaling cascade involving integrins, Rho GTPases, and membrane-type matrix metalloproteinases (MT-MMPs). [8][9][10][11][12] These signaling events stimulate EC intracellular vacuole formation and coalescence that controls EC lumen formation in vitro and in vivo. 8,10,12 A variety of integrins have been described to be relevant in regulating angiogenesis and tube formation including both ␤1 and ␣v integrins. The relevance of any particular integrin appears to be primarily dependent on the matrix environment (eg, adult, embryonic, wound, tumor) where the EC tube morphogenic process takes place. 3,9,[15][16][17][18][19] Extracellular matrix (ECM) proteolysis is thought to be an important step in how cells move through 3D matrix environments [20][21][22][23][24][25][26][27] and has been implicated in vessel for...

Hic-5 mediates endothelial sprout initiation by regulating a key surface metalloproteinase

“…S2A). Endothelial lumen formation is a crucial aspect of endothelial morphogenesis and tubulogenesis (Davis et al, 2011), and MT1-MMP is required for both successful lumen formation and endothelial cell sprouting into collagen matrices (Chun et al, 2004;Stratman et al, 2009;Sacharidou et al, 2010). S1P has been shown to stimulate phosphorylation of MT1-MMP at Y573 (Nyalendo et al, 2007).…”

“…MT1-MMP is a membrane-anchored metalloproteinase that cleaves matrix proteins in close proximity to the cell surface, thus maintaining overall matrix integrity to support invading structures (Werb, 1997;Hiraoka et al, 1998;Murphy and Gavrilovic, 1999;Hotary et al, 2000). MT1-MMP has been shown to direct endothelial sprouting and lumen formation, as well as tumor cell invasion (Hiraoka et al, 1998;Yana and Seiki, 2003;Chun et al, 2004;Stratman et al, 2009). Following translation, the pro-domain of MT1-MMP is Endothelial cells cultured in 10-cm dishes were stimulated with nothing (CON) or 1 μM S1P for 1 h. Immunoprecipitations (IP) were performed using monoclonal FAK antibodies or isotype (IgG) controls.…”

“…Our results indicate that Hic-5 contributes to these events in endothelial cells by bridging MT1-MMP and FAK. FAK is a well-known regulator of angiogenesis (Peng et al, 2004;Shen et al, 2005), as is MT1-MMP (Zhou et al, 2000;Chun et al, 2004;Stratman et al, 2009). Thus, defining molecular events that coordinate and regulate the activities of these key proteins will further refine our understanding of new blood vessel growth.…”

“…MT1-MMP is also required for lumen formation in vitro. Overexpression of wild-type MT1-MMP increases lumen formation, whereas MT1-MMP small interfering RNA (siRNA) blocks invasion and lumen formation (Stratman et al, 2009). Finally, MT1-MMP gene disruption causes abnormalities in osteogenesis, FGF-induced angiogenesis and collagen turnover, leading to reduced neovessel formation, dwarfism, arthritis and premature death in knockout mice (Holmbeck et al, 1999;Zhou et al, 2000).…”

Tips, Stalks, Tubes: Notch-Mediated Cell Fate Determination and Mechanisms of Tubulogenesis during Angiogenesis

“…Capillary-like sprouts form and migrate into the fibrin clot, ultimately elongating, anastomosing, and forming quantifiable lumens (Nakatsu et al 2003;Nakatsu et al 2007). Another in vitro assay uses both pericytes and endothelial cells to form a patent vessel in collagen, with evidence of basement membrane formation (Stratman et al 2009). In this assay, endothelial cells and pericytes are cocultured in a three-dimensional collagen gel with special medium.…”