SUMMARYCerebral cavernous malformation (CCM) is a common vascular dysplasia that affects both systemic and CNS blood vessels. Loss of function mutations in the CCM2 gene cause CCM. Here we show that targeted disruption of Ccm2 in mice results in failed lumen formation and early embryonic death through an endothelial cell autonomous mechanism. We demonstrate that CCM2 regulates endothelial cytoskeletal architecture, cell-cell interactions and lumen formation. Heterozygosity at Ccm2, a genotype equivalent to human CCM, results in impaired endothelial barrier function. Because our biochemical studies indicate that loss of CCM2 results in activation of RHOA GTPase, we rescued the cellular phenotype and barrier function in heterozygous mice using simvastatin, a drug known to inhibit Rho GTPases. These data offer the prospect for pharmacologic treatment of a human vascular dysplasia using a widely available and safe drug.

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...

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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.