Microvascular networks support metabolic activity and define microenvironmental conditions within tissues in health and pathology. Recapitulation of functional microvascular structures in vitro could provide a platform for the study of complex vascular phenomena, including angiogenesis and thrombosis. We have engineered living microvascular networks in three-dimensional tissue scaffolds and demonstrated their biofunctionality in vitro. We describe the lithographic technique used to form endothelialized microfluidic vessels within a native collagen matrix; we characterize the morphology, mass transfer processes, and long-term stability of the endothelium; we elucidate the angiogenic activities of the endothelia and differential interactions with perivascular cells seeded in the collagen bulk; and we demonstrate the nonthrombotic nature of the vascular endothelium and its transition to a prothrombotic state during an inflammatory response. The success of these microvascular networks in recapitulating these phenomena points to the broad potential of this platform for the study of cardiovascular biology and pathophysiology.tissue engineering | regenerative medicine | microfluidics | cancer | blood T he microvasculature is an extensive organ that mediates the interaction between blood and tissues. It defines the biological and physical characteristics of the microenvironment within tissues and plays a role in the initiation and progression of many pathologies, including cancer (1) and cardiovascular diseases (2, 3). Conventional planar cultures fail to recreate the in vivo physiology of the microvasculature with respect to three-dimensional (3D) geometry (lumens and axial branching points), and interactions of endothelium with perivascular cells, extracellular tissue and blood flow (4). Studies of the microvasculature in vivo allow only limited control of physical, chemical, and biological parameters influencing the microvasculature and present challenges with respect to observation (5). In vitro cultures that produce tubular vessels within 3D matrices will aid in elucidation of the roles of the microvasculature in health and disease. Important progress has been made toward this goal: Biologically derived or synthetic materials have been used to generate macrovessel tubes (6) and endothelialized microtubes (7); cellular self-assembly has been used to generate random microvasculature (8); microfabrication has been used to define complex geometries in hydrogels at the micro-scale (9); and distributions of cells and biochemical factors within 3D scaffolds (10). Of particular note, the group of Tien has pioneered the use of collagen to template the growth of vascular endothelium (7, 11) and demonstrated appropriate permeability (7), response to cyclic AMP (12), and differential properties as a function of the luminal shear stress and composition of the medium (13). Nonetheless, prior methodologies have been unable to produce endothelialized networks that can undergo substantial remodeling via angiogenesis; elucidate the ...

A central tenet of fibrinolysis is that tissue plasminogen activator-dependent (t-PA- dependent) conversion of plasminogen to active plasmin requires the presence of the cofactor/substrate fibrin. However, previous in vitro studies have suggested that the endothelial cell surface protein annexin II can stimulate t-PA-mediated plasminogen activation in the complete absence of fibrin. Here, homozygous annexin II-null mice displayed deposition of fibrin in the microvasculature and incomplete clearance of injury-induced arterial thrombi. While these animals demonstrated normal lysis of a fibrin-containing plasma clot, t-PA-dependent plasmin generation at the endothelial cell surface was markedly deficient. Directed migration of annexin II-null endothelial cells through fibrin and collagen lattices in vitro was also reduced, and an annexin II peptide mimicking sequences necessary for t-PA binding blocked endothelial cell invasion of Matrigel implants in wild-type mice. In addition, annexin II-deficient mice displayed markedly diminished neovascularization of fibroblast growth factor-stimulated cornea and of oxygen-primed neonatal retina. Capillary sprouting from annexin II-deficient aortic ring explants was markedly reduced in association with severe impairment of activation of metalloproteinase-9 and -13. These data establish annexin II as a regulator of cell surface plasmin generation and reveal that impaired endothelial cell fibrinolytic activity constitutes a barrier to effective neoangiogenesis.

This protocol describes how to form a 3D cell culture with explicit, endothelialized microvessels. The approach leads to fully enclosed, perfusable vessels in a bioremodelable hydrogel (type I collagen). The protocol uses microfabrication to enable user-defined geometries of the vascular network and microfluidic perfusion to control mass transfer and hemodynamic forces. These microvascular networks (μVNs) allow for multiweek cultures of endothelial cells or cocultures with parenchymal or tissue cells in the extra-lumen space. The platform enables real-time fluorescence imaging of living engineered tissues, in situ confocal fluorescence of fixed cultures and transmission electron microscopy (TEM) imaging of histological sections. This protocol enables studies of basic vascular and blood biology, provides a model for diseases such as tumor angiogenesis or thrombosis and serves as a starting point for constructing prevascularized tissues for regenerative medicine. After one-time microfabrication steps, the system can be assembled in less than 1 d and experiments can run for weeks.

Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19 ؊/؊ animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects.ADAMs (mnemonic for a disintegrin and metalloprotease) are membrane-anchored glycoproteins with key roles in fertilization, neurogenesis, angiogenesis, Alzheimer's disease, and the release of proteins such as epidermal growth factor (EGF) receptor ligands and tumor necrosis factor family members from the plasma membrane (3,4,17,37,39,41). ADAM19 (also referred to as meltrin ␤) was initially identified in muscle cells and was later found to be expressed in several other tissues, most prominently in heart, lung, and bone (18, 27, 52), during dendritic cell differentiation (13) and Notch-induced T-cell maturation (9). The catalytic activity of ADAM19 towards candidate substrates has been explored by overexpression in cells and by purifying recombinantly expressed soluble forms of the entire ectodomain or the pro-and metalloprotease domains (7,42,49,53). Overexpressed ADAM19 enhances ectodomain shedding of two of several splice variants of neuregulin I-␤ (42), a ligand for the ErbB family of receptor tyrosine kinases (11). Furthermore, overexpression of ADAM19 increases ectodomain release of tumor necrosis factor-related activation-induced cytokine (TRANCE, also referred to as osteoprotegerin-ligand [OPGL]) (7), a protein with important roles in osteoclast differentiation, dendritic cell survival, and mammary gland development (12,25,28).In light of the high expression of ADAM19 in heart and bone and its ability to cleave TRANCE as well as splice variants of neuregulin I-␤, we were interested in evaluating the function of ADAM19 in mice, with an emphasis on its role in heart and bone development. Here we present an analysis of mice lacking functional ADAM19 (adam19 Ϫ/Ϫ mice). MATERIALS AND METHODS Generation of adam19؊/؊ mice. adam19 ϩ/Ϫ mice were generated by the SloanKettering Institute transgenic facility by following standard procedures using stem cells with a secretory gene trap insertion in ADAM19 (30). All mice evaluated in this study were of mixed genetic background (129Sv/C57BL6), and morphological and hist...

Oxygen status and tissue dimensionality are critical determinants of tumor angiogenesis, a hallmark of cancer and an enduring target for therapeutic intervention. However, it is unclear how these microenvironmental conditions interact to promote neovascularization, due in part to a lack of comprehensive, unbiased data sets describing tumor cell gene expression as a function of oxygen levels within three-dimensional (3D) culture. Here, we utilized alginate-based, oxygen-controlled 3D tumor models to study the interdependence of culture context and the hypoxia response. Microarray gene expression analysis of tumor cells cultured in 2D versus 3D under ambient or hypoxic conditions revealed striking interdependence between culture dimensionality and hypoxia response, which was mediated in part by pro-inflammatory signaling pathways. In particular, interleukin-8 (IL-8) emerged as a major player in the microenvironmental regulation of the hypoxia program. Notably, this interaction between dimensionality and oxygen status via IL-8 increased angiogenic sprouting in a 3D endothelial invasion assay. Taken together, our data suggest that pro-inflammatory pathways are critical regulators of tumor hypoxia response within 3D environments that ultimately impact tumor angiogenesis, potentially providing important therapeutic targets. Furthermore, these results highlight the importance of pathologically relevant tissue culture models to study the complex physical and chemical processes by which the cancer microenvironment mediates new vessel formation.