We show that endothelial cell (EC)-generated vascular guidance tunnels (ie, matrix spaces created during tube formation) serve as conduits for the recruitment and motility of pericytes along EC ablumenal surfaces to facilitate vessel maturation events, including vascular basement membrane matrix assembly and restriction of EC tube diameter. During quail development, pericyte recruitment along microvascular tubes directly correlates with vascular basement membrane matrix deposition. Pericyte recruitment to EC tubes leads to specific induction of fibronectin and nidogen-1 (ie, matrixbridging proteins that link together basement membrane components) as well as perlecan and laminin isoforms. Coincident with these events, up-regulation of integrins, ␣ 5 ␤ 1 , ␣ 3 ␤ 1 , ␣ 6 ␤ 1 , and ␣ 1 ␤ 1 , which bind fibronectin, nidogens, laminin isoforms, and collagen type IV, occurs in EC-pericyte cocultures, but not EC-only cultures. Integrin-blocking antibodies to these receptors, disruption of fibronectin matrix assembly, and small interfering RNA suppression of pericyte tissue inhibitor of metalloproteinase (TIMP) - IntroductionConsiderable interest has focused on determining how support cells such as pericytes affect the vasculature during development and in various disease states. 1-3 An important step in vascular morphogenesis is the recruitment of pericytes, which, in conjunction with endothelial cells (ECs), establish conditions to facilitate tube stabilization. 2,4-11 EC factors such as platelet-derived growth factor-BB play a critical role in these events, and failure to recruit pericytes during development leads to vascular instability and regression. 4,[12][13][14] Thus, abnormalities in EC-pericyte interactions lead to embryonic death due to failures in vascular remodeling and stabilization. 2,11,12 Recently, we reported that pericyte recruitment to EC tubes induced stabilization by affecting the production and function of EC-derived tissue inhibitor of metalloproteinase (TIMP)-2 and pericyte-derived TIMP-3, which led to inhibition of both tube regression and morphogenic events through blockade of particular matrix metalloproteinases (MMPs). 15 The molecular mechanisms controlling how pericytes affect vascular tube stabilization are being elucidated and include the identification of key growth factors regulating these events, such as angiopoietin-1, vascular endothelial growth factor (VEGF), and transforming growth factor-␤ (TGF-␤), signaling pathways involving Notch and Ephrins, as well as the presentation of MMP inhibitors such as [7][8][9]11,[15][16][17][18][19][20][21][22][23] Recent work from our laboratory has identified a key regulatory step in vessel formation, which is a requirement for membrane type 1 (MT1)-MMP in both EC lumen and vascular guidance tunnel formation. 24 Vascular guidance tunnels are generated in conjunction with EC tube morphogenesis and represent physical spaces throughout the matrix that serve as conduits for tube assembly, remodeling, and recruitment of other cell types such as pericy...

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

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...

Recently, we reported a novel system whereby human pericytes are recruited to endothelial cell (EC)-lined tubes in 3-dimensional (3D) extracellular matrices to stimulate vascular maturation including basement membrane matrix assembly. Through the use of this serum-free, defined system, we demonstrate that pericyte motility within 3D collagen matrices is dependent on the copresence of ECs.

No abstract

Many studies reveal a fundamental role for extracellular matrix-mediated signaling through integrins and Rho GTPases as well as matrix metalloproteinases (MMPs) in the molecular control of vascular tube morphogenesis in three-dimensional (3D) tissue environments. Recent work has defined an EC lumen signaling complex of proteins that controls these vascular morphogenic events. These findings reveal a signaling interdependence between Cdc42 and MT1-MMP to control the 3D matrix-specific process of EC tubulogenesis. The EC tube formation process results in the creation of a network of proteolytically-generated vascular guidance tunnels in 3D matrices that are utilized to remodel EC-lined tubes through EC motility and could facilitate processes such as flow-induced remodeling and arteriovenous EC sorting and differentiation. Within vascular guidance tunnels, key dynamic interactions occur between endothelial cells (ECs) and pericytes to affect vessel remodeling, diameter, and vascular basement membrane matrix assembly, a fundamental process necessary for endothelial tube maturation and stabilization. Thus, the EC lumen and tube formation mechanism coordinates the concomitant establishment of a network of vascular tubes within tunnel spaces to allow for flow responsiveness, EC-mural cell interactions, and vascular extracellular matrix assembly to control the development of the functional microcirculation.

Here, we define an endothelial cell (EC) lumen signaling complex involving Cdc42, Par6b, Par3, junction adhesion molecule (Jam)-B and Jam-C, membrane type 1-matrix metalloproteinase (MT1-MMP), and integrin ␣ 2 ␤ 1 , which coassociate to control human EC tubulogenesis in 3D collagen matrices. Blockade of both Jam-B and Jam-C using antibodies, siRNA, or dominant-negative mutants completely interferes with lumen and tube formation resulting from a lack of Cdc42 activation, inhibition of Cdc42-GTP-dependent signal transduction, and blockade of MT1-MMP-dependent proteolysis. This process requires interdependent Cdc42 and MT1-MMP signaling, which involves Par3 binding to the Jam-B and Jam-C cytoplasmic tails, an interaction that is necessary to physically couple the components of the lumen signaling complex. MT1-MMP proteolytic activity is necessary for Cdc42 activation during EC tube formation in 3D collagen matrices but not on 2D collagen surfaces, whereas Cdc42 activation is necessary for MT1-MMP to create vascular guidance tunnels and tube networks in 3D matrices through proteolytic events. This work reveals a novel interdependent role for Cdc42-dependent signaling and MT1-MMP-dependent proteolysis, a process that occurs selectively in 3D collagen matrices and that requires EC lumen signaling complexes, to control human EC tubulogenesis during vascular morphogenesis. (Blood. 2010;115(25):5259-5269) IntroductionRecent work has lead to an increased understanding of how endothelial and epithelial cells make lumens and tubes in 3D extracellular matrices. [1][2][3][4][5][6][7][8][9] Key regulators of lumen formation include Cdc42, which was first shown to regulate this process in endothelial cells (ECs), [10][11][12] and later in epithelial cells. 13,14 Components of the cell polarity machinery including Par3, Par6, and PKC control lumen formation of both cell types. 6,11,[13][14][15] Furthermore, we recently reported that Cdc42 activates a signaling cascade involving PKC⑀, Pak2, Pak4, Src, Yes, B-Raf, C-Raf, and Erk1/2 to control this process. 12,16 In addition, EC-directed cellsurface proteolytic events through membrane type 1-matrix metalloproteinase (MT1-MMP) 17,18 controls EC lumen and vascular guidance tunnel formation in 3D collagen matrices. 19,20 A key question that has remained unresolved is how Cdc42-dependent signaling and MT1-MMP-dependent proteolysis are functionally coupled to regulate EC tube formation. 2 Recent studies have revealed that MT1-MMP directs 3D matrix-specific events in relationship to tumor motility, cellular differentiation, and morphogenesis. 2,21 We have shown that both tumor cell and EC invasion of 3D collagen matrices requires MT1-MMP, but not motility on 2D collagen substrates. 19,22 Interestingly, adipocyte differentiation occurs in an MT1-MMP-dependent manner in 3D matrices but not on 2D matrix surfaces, 23 and MT1-MMP controls the 3D-specific process of EC lumen and tube formation. 18,19 Thus, MT1-MMP is functionally linked to critical cellular events that specifically occur in 3D...

Extracellular matrix (ECM) synthesis and deposition surrounding the developing vasculature is critical for vessel remodeling and maturation events. Although the basement membrane is an integral structure underlying endothelial cells (ECs), few studies, until recently, have been performed to understand its formation in this context. In this review, we highlight new data demonstrating a co-requirement for ECs and pericytes to properly deposit and assemble vascular basement membranes during morphogenic events. In EC only cultures or under conditions whereby pericyte recruitment is blocked, there is a lack of basement membrane assembly, decreased vessel stability (with increased susceptibility to pro-regressive stimuli) and increased EC tube widths (a marker of dysfunctional EC-pericyte interactions). ECs and pericytes both contribute basement membrane components and, furthermore, both cells induce the expression of particular components as well as integrins that recognize them. The EC-derived factors, platelet derived growth factor-BB (PDGF-BB) and heparin binding-epidermal growth factor (HB-EGF), are both critical for pericyte recruitment to EC tubes and concomitant vascular basement membrane formation in vitro and in vivo. Thus, heterotypic EC-pericyte interactions play a fundamental role in vascular basement membrane matrix deposition, a critical tube maturation event that is altered in key disease states such as diabetes and cancer.